Platinum compounds that inhibit constitutive STAT3 signaling and induce cell cycle arrest and apoptosis of malignant cells

ABSTRACT

The subject invention concerns a compound and compositions having activity as an inhibitor of Stat3 protein and methods of using the compound and compositions. In one embodiment, a compound of the invention has the structure shown in formula I, formula II, or formula III. The subject invention also concerns methods of using the compounds and compositions of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. applicationSer. No. 14/354,400, filed Apr. 25, 2014, which is the National Stage ofInternational Application No. PCT/US2012/062272, filed Oct. 26, 2012,which claims the benefit of U.S. Provisional Application Ser. No.61/551,737, filed Oct. 26, 2011, each of which is hereby incorporated byreference herein in its entirety, including any figures, tables, nucleicacid sequences, amino acid sequences, and drawings.

GOVERNMENT SUPPORT

This invention was made with government support under grant numberCA55652 awarded by the National Cancer Institute. The government hascertain rights in the invention.

The Sequence Listing for this application is labeled “2GE6844.TXT” whichwas created on Nov. 19, 2015 and is 1.26 KB. The entire contents of thesequence listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Cellular responses to growth factors and cytokines are characterized bythe activation of signal transduction pathways, including the SignalTransducer and Activator of Transcription (STAT) family of cytoplasmictranscription factors (Darnell et al., 1994; Schindler et al., 1995;Darnell, 1997; Stark et al., 1998). Activation of STAT proteins isinitiated upon their tyrosine phosphorylation, a key event in theformation of phosphotyrosine-SH2 (pTyr-SH2) interactions and thedimerization between two STAT monomers. In turn, dimers of STATstranslocate to the nucleus and bind to specific DNA-response elements,thereby inducing the expression of genes essential for cellularresponses. Normal physiological functions of STATs include regulation ofcell proliferation, differentiation, development and apoptosis (reviewedin (Bromberg et al., 1996; Fukada et al., 1996; Kotenko et al., 2000;Smithgall et al., 2000; Hirano et al., 2000; Akira, 2000)).

In contrast to the tightly-regulated normal STAT signaling, constitutiveactivation of STAT proteins is frequently observed in human tumors(Turkson et al., 1998; Bromberg et al., 1998) and has been linked totumor progression. Persistent activation of one STAT family member,Stat3, is detected in breast cancer, prostate cancer, head and necksquamous cell carcinoma, as well as in lymphomas and leukemias (Garciaet al., 1997; Nielsen et al., 1997; Catlett-Falcone et al., 1999;Nielsen et al., 1999; Bromberg, 2000; Grandis et al., 2000; Garcia etal., 2001; Epling-Burnette et al., 2001), reviewed in (Bowman et al.,2000a; Turkson et al., 2000; Song et al., 2000; Coffer et al., 2000; Linet al., 2000; Buettner et al., 2002; Yu et al., 2004; Turkson, 2004a).In malignant cell lines and tumors that harbor constitutively-activeStat3, studies also reveal overexpression of Stat3-regulated genesencoding the anti-apoptotic proteins Bcl-xL and Mcl-1, the cell cycleregulators, Cyclin D1 and c-Myc, the angiogenesis factor, VEGF, as wellas altered expression of immune-modulatory factors (Catlett-Falcone etal., 1999; Nielsen et al., 1999; Grandis et al., 2000; Epling-Burnetteet al., 2001; Bowman et al., 2000b; Niu et al., 2002; Wang et al.,2004a). These abnormal gene expression changes contribute todysregulated cell cycle progression, survival and angiogenesis, and torepressed host immune functions (reviewed in (Yu et al., 2004; Turkson,2004b)). Thus, inhibition of abnormal Stat3 signaling is sufficient torepress the induction of these genes, resulting in cell cycle arrest andapoptosis of malignant cells (Catlett-Falcone et al., 1999; Grandis etal., 2000; Epling-Burnette et al., 2001; Niu et al., 1999),sensitization of tumor cells to chemotherapy-induced apoptosis (Oshiroet al., 2001), anti-tumor immune responses (Wang et al., 2004a), andtumor regression (Niu et al., 1999). Small-molecule inhibitors of Stat3,therefore, have the potential to impact tumors that harborconstitutively-active Stat3 with significant clinical benefits.

Previous studies have implicated signal transduction pathways in theantitumor activity of platinum complexes. Evidence shows that Cisplatinmight modulate the mitogen-activated protein kinase family and thePI-3-kinase/Akt pathway (Sanchez-Perez et al., 1998; Persons et al.,1999; Bose, 2002; Siddik, 2003). Platinum complexes that inhibit Stat3signaling and induce tumor regression have previously been reported(Turkson et al., 2004b).

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns a compound and compositions havingactivity as an inhibitor of Stat3 protein and methods of using thecompound and compositions. In one embodiment, a compound of theinvention, designated herein as PLATINUM-401, has the structure shown informula I:

The compound in formula I is designated herein as Platinum-401. Analysesof in vitro DNA-binding activity and transcriptional regulation indicatethat PLATINUM-401 interacts directly with Stat3, thereby inhibitingStat3 binding to a consensus DNA response element and Stat3transcriptional activity. Inhibition of constitutively-active Stat3 inmalignant cells by PLATINUM-401 suppresses the induction ofStat3-regulated genes, including Bcl-xL and Cyclin D1. Studies inv-Src-transformed fibroblasts as well as in human and mouse tumor celllines that harbor constitutive Stat3 activity reveal a G₀/G₁ cell cyclearrest and apoptosis following treatment with PLATINUM-401, whichcorrelate with the inhibition of aberrant Stat3 signaling.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E. Inhibition of in vitro Stat3 DNA-binding activity by aplatinum complex. Nuclear extracts or cell lysates containing activatedStat1, Stat3 and Stat5, or E2F1 prepared from NIH3T3/hEGFR that arestimulated with EGF or Sf-9 insect cells that are infected withbaculovirus expressing Stat1, Stat3, Stat5, or E2F1, respectively, weretreated with or without the indicated concentrations of platinumcomplex, PLATINUM-401 or Cisplatin, for 30 min at room temperature priorto incubation with radiolabeled oligonucleotide probes, and subjected toEMSA analysis. (FIG. 1A) (i) Stat1 and Stat3 binding activities to hSIEprobe, (ii) Stat1 and Stat5 binding activities to MGFe probe, and (iii)plot of % oligonucleotide probe-STAT:STAT complexes versus concentrationof PLATINUM-401. Insert, IC₅₀ values for the inhibition of STAT:STATDNA-binding activity; (FIG. 1B) (i) E2F1 binding activity to thedihydrofolate reductase promoter oligonucleotide probe, and (ii) Stat1and Stat3 binding activities to hSIE probe; (FIG. 1C) PLATINUM-401effect on the in vitro DNA-binding activity of Stat3, (i) 3-30 min afterbinding, or in the presence and absence of (ii) inactive Stat3 monomer,(iii) inactive Stat1 monomer, (iv) inactive Stat5 monomer, or (v) E2F1protein; (FIG. 1D) Stat1 and Stat3 binding to PLATINUM-401-treated anduntreated (DMSO) radiolabeled hSIE probe; and (FIG. 1E) structuralformula of PLATINUM-401. Positions of STAT:STAT-DNA complexes in gel arelabeled. In (A) to (C), the control lanes represent DMSO(vehicle)-treatment.

FIGS. 2A-2E. Kinetics of PLATINUM-401-mediated inhibition of in vitroStat3 DNA-binding activity. Cell lysates containing activated Stat3 wereincubated with radiolabeled hSIE probe for 30 min at room temperature inthe presence or absence of PLATINUM-401 and then subjected to EMSAanalysis. (FIG. 2A) DNA-binding activities for different Stat3 proteinamounts in the presence of increasing concentrations of PLATINUM-401;(FIG. 2B) Levels of Stat3 binding to increasing amounts of hSIEoligonucleotide probe in the absence (control) and presence ofPLATINUM-401; (FIG. 2C) Levels of Stat3 binding to hSIE oligonucleotideprobe in the presence of increasing concentrations of PLATINUM-401;(FIG. 2D) Plots of hSIE-Stat3:Stat3 complex versus levels of hSIEoligonucleotide probe under different concentrations (0.3-2 μM) ofPLATINUM-401; (FIG. 2E) Lineweaver-Burke analysis (double reciprocalplot) of hSIE-Stat3 complex versus levels of hSIE under differentconcentrations (0.3-2 μM) of PLATINUM-401. Positions of Stat3:Stat3-DNAcomplexes in gels are labeled.

FIGS. 3A-3D. Inhibition of Stat3-mediated gene expression byPLATINUM-401. (FIG. 3A) and (FIG. 3B) v-Src-transformed mousefibroblasts that stably express Stat3-dependent (NIH3T3/v-Src/pLucTKS3)and Stat3-independent (NIH3T3/v-Src/pRLSRE) luciferase reporters orβ-galactosidase (β-gal), as well as normal mouse fibroblast (NIH3T3)transiently transfected with pLucTKS3, pGL2-VEGF-Luc, NFkB-Luc orpLucSRE together with or without v-Src were treated with or withoutPLATINUM-401 for 48 h. Cytosolic extracts were then prepared from cellsfor luciferase and 8-gal activities measurements. Values are the meansand S.D. of three to five independent assays; (FIG. 3C) and (FIG. 3D)Nuclear extracts or whole cell lysates were prepared fromIL-6-stimulated normal mouse fibroblasts (NIH3T3) or theirv-Src-transformed counterpart (NIH3T3/v-Src) that are treated with orwithout PLATINUM-401 for different times. Samples of equal totalproteins were then subjected to in vitro DNA-binding activity and EMSAanalysis or for SDS-PAGE and Western blot analysis for phosphorylatedand total Stat3.

FIG. 4. Abrogation of Stat3-dependent viral Src transformation. ViralSrc-transformed fibroblasts (NIH3T3/v-Src) and their Ras-transformedcounterparts (NIH3T3/v-Ras) were seeded in soft agar and growing cellswere treated every 2-3 days with or without the indicated concentrationsof PLATINUM-401 until large colonies were evident. Number of colonies ofcells in soft agar were counted and expressed as % of control(non-treated) cells. Values are the mean and S.D. of the threeindependent assays.

FIGS. 5A and 5B. Evaluation for effects of PLATINUM-401 on cellularconstitutive Stat3 activation and cell proliferation. Normal ormalignant cells were treated with or without PLATINUM-401 and nuclearextracts were prepared for Stat3 DNA-binding activity assay with hSIEprobe, or cells were processed for nuclear Ki67 immunohistochemistry.(FIG. 5A) EMSA analysis of Stat3 DNA-binding activity; (FIG. 5B)Graphical representations of quantified nuclear staining of Ki-67proliferation index. Stat3:Stat3-DNA complexes in gels are labeled. TheKi-67 proliferation indexes were calculated as the percent positivetumor cells relative to the total number of cells. Ki67 values arerepresentative of 3 independent assays.

FIG. 6. Analysis of relative cellular DNA content using BrdU labelingand flow cytometry. Relative DNA content of human breast cancer celllines following treatment with or without PLATINUM-401 was analyzed byBrdU incorporation and flow cytometry. The population of cellsdetermined from the relative DNA content is shown in each panel for eachtreatment condition. Results are the representative of 3 independentdeterminations.

FIGS. 7A-7C. TUNEL analysis of PLATINUM-401-mediated apoptosis. (FIG.7A) Normal NIH3T3 fibroblasts and their v-Src-transformed counterparts(NIH3T3/v-Src), human breast carcinoma cell lines (MDA-MB-453,MDA-MB-435, MDA-MB-468, and MDA-MB-231), human non-small cell lungcancer cell line (A549), human prostate cancer cell line (DU145),multiple myeloma 5TGM1 (mouse) and U266 (human) cell lines, mousemelanoma cell line (B16) and human pancreatic cancer cell line (Panc1)were all treated with or without PLATINUM-401 for 48 h and analyzed byTUNEL for DNA damage. For each cell line, the activated Stat3 status isindicated as (−), no constitutively-active Stat3 and (+),constitutively-active Stat3 (see FIG. 5A). Data are representative of 3independent determinations; (FIG. 7B) Human breast cancer cell lines(MDA-MB-453, MDA-MB-468 and MDA-MB-435) were transfected with or withoutStat3β or Stat3 antisense (Stat3AS), or were treated with or withoutStat3 peptidomimetic inhibitor, ISS 610 (1 mM) or PLATINUM-401 (5 μM)).Forty-eight hours afterwards, cells were harvested and processed forTUNEL analysis; (FIG. 7C) The viral Src-transformed NIH3T3/v-Srcfibroblasts were transfected with or without wild-type Stat3 (pRc/CMVStat3 Flag) and treated with or without 5 μM PLATINUM-401 for 36 h.Cells were subsequently harvested for nuclear extracts preparation andStat3 DNA-binding assay in vitro with EMSA analysis (left panel), orwere processed for TUNEL analysis (right panel).

FIGS. 8A and 8B. Inhibition of Cyclin D1 and Bcl-xL induction byPLATINUM-401. Viral Src-transformed fibroblasts (NIH3T3/v-Src) and humanbreast cancer cell line MDA-MB-435 that contain constitutively-activatedStat3 were treated with or without platinum complex for 48 h. Cells wereprocessed for immunohistochemistry, or cell lysates were prepared fromcells and subjected to 5% PAGE and Western blot analysis, as indicatedin “Materials and Methods.” (FIG. 8A) Detection of Cyclin D1; (FIG. 8B)Detection of Bcl-xL. Positions of Cyclin D1 and Bcl-xL proteins areshown. Beta-actin levels are shown for normalizing for equal totalprotein. Data are representative of 3 independent determinations.

FIG. 9. STAT3 DNA-binding activity/EMSA assay in vitro and the effectsof compounds. Nuclear extracts or cell lysates containing activatedStat3 were treated with or without the indicated concentrations of NSC251168, platinum complex K2PtC16, PLATINUM-401 (supplied by Dr.Turkson), PLATINUM-401 (supplied by Dr. Lawrence as described in Example10 herein), RPM1581 (free base) (supplied by Dr. Lawrence as describedin Example 10 herein and also designated as YL5-108), or RPM1581-2HCl(salt form) (supplied by Dr. Lawrence), for 30 min at room temperatureprior to incubation with radiolabeled oligonucleotide probes, andsubjected to EMSA analysis.

FIG. 10. Inhibition of Stat3 activation in tumor cells. In each case,NIH3T3/y-Src or human SKOV3 cells were treated with 0, 10, or 30micromolar concentrations of NSC 251168, platinum complex K2PtC16, orPLATINUM-401 (supplied by Dr. Lawrence as described in Example 10herein) drug for 24 hr, and Stat3 activation assayed by EMSA.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is an oligonucleotide that can be used as described herein.

SEQ ID NO:2 is an oligonucleotide that can be used as described herein.

SEQ ID NO:3 is an oligonucleotide that can be used as described herein.

SEQ ID NO:4 is an oligonucleotide that can be used as described herein.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention concerns compounds and compositions disclosedherein having activity as inhibitors of Stat3 protein and methods ofusing the compounds and compositions. In one embodiment, a compound ofthe invention, designated herein as PLATINUM-401, has the structureshown in formula I:

In another embodiment, a compound of the invention, designated herein asRPM 1581, has the structure shown in formula II (as a free base) or informula III (as a salt):

In one embodiment, the compound of formula III is provided as thechloride salt.

The subject invention also concerns compositions comprising a compoundof formula I, or formula II, or formula III, and a carrier, buffer,and/or adjuvant.

The subject invention also concerns methods for treating a person oranimal having a disorder or condition associated with aberrant orexcessive Stat3 activity or interaction in a cell or decreased apoptosisof a cell, or a disorder or condition associated with inhibition ordownregulation of apoptosis of a cell. In one embodiment, the disorderor condition is an oncological disorder or condition. In one embodiment,a person or animal is administered an effective amount of an inhibitorcompound or composition of this invention. In one embodiment, thecompound is the compound designated herein as PLATINUM-401 (formula I).In another embodiment, the compound is a chloroplatinic acid (H₂PtCl₆ orK₂PtCl₆, etc.). In still a further embodiment, the compound is thecompound designated herein as RPM 1581 which can be in a free base(formula II) or salt form (e.g., formula III). In one embodiment,compounds and compositions of the invention can be used in combinationwith other Stat3 inhibitors, including, but not limited to, NSC 74859(also known as S3I-201; Siddiquee et al. (2007)), protein inhibitor ofactivated Stat3 (PIAS3) (Ueki et al. (1999)), WP1066 (Farzana Hussain etal. (2007)), or STA-21 (Song et al. (2005)). Oncological disordersinclude, but are not limited to, prostate cancer, head and neck squamouscell carcinoma, lymphomas, leukemias, and breast cancer.

The subject invention also concerns methods of inducing apoptosis in acell. In one embodiment, a cell is contacted with an effective amount ofan inhibitor compound or composition of this invention. In oneembodiment, the compound is the compound designated herein asPLATINUM-401 (formula I). In another embodiment, the compound is achloroplatinic acid (H₂PtCl₆ or K₂PtCl₆). In still a further embodiment,the compound is the compound designated herein as RPM 1581 which can bein a free base (formula II) or salt form (e.g., formula III). Cells canbe any animal cell, such as a mammalian cell. Cells can be any mammaliancell, such as a human cell, canine cell, feline cell, or equine cell. Inone embodiment the cell is a tumor cell, a cancer cell or a transformedcell. In one embodiment, the tumor or cancer cell is a breast cancercell, prostate cancer cell, head and neck squamous cell carcinoma,lymphoma cell, leukemia cell, multiple myeloma cell, glioma cell,non-small cell lung cancer cell, melanoma cell, gastrointestinal stromaltumor cell, renal cell carcinoma, esophageal cell carcinoma, ovariancancer cell, cervical cancer cell, or gastric cancer cell. In oneembodiment, compounds and compositions of the invention can be used incombination with other Stat3 inhibitors, including, but not limited to,NSC 74859, PIAS3, WP1066, or STA-21.

The subject invention also concerns methods for inhibiting Stat3interaction in a cell. In one embodiment, a cell is contacted with aneffective amount of an inhibitor compound or composition of thisinvention. In one embodiment, the compound is the compound designatedherein as PLATINUM-401 (formula I). In another embodiment, the compoundis a chloroplatinic acid (H₂PtCl₆ or K₂PtCl₆). In still a furtherembodiment, the compound is the compound designated herein as RPM 1581which can be in a free base (formula II) or salt form (e.g., formulaIII). Cells can be any animal cell, such as a mammalian cell. Cells canbe any mammalian cell, such as a human cell, canine cell, feline cell,or equine cell. In one embodiment the cell is a tumor cell, a cancercell or a transformed cell. In one embodiment, the tumor or cancer cellis a breast cancer cell, prostate cancer cell, head and neck squamouscell carcinoma, lymphoma cell, leukemia cell, multiple myeloma cell,glioma cell, non-small cell lung cancer cell, melanoma cell,gastrointestinal stromal tumor cell, renal cell carcinoma, esophagealcell carcinoma, ovarian cancer cell, cervical cancer cell, or gastriccancer cell. In one embodiment, compounds and compositions of theinvention can be used in combination with other Stat3 inhibitors,including, but not limited to, NSC 74859, PIAS3, WP1066, or STA-21.

In some embodiments of the methods of the invention, the method furthercomprises a step to determine whether the disorder or condition isassociated with aberrant or excessive Stat3 activity or interaction, orto verify that the cell aberrantly or constitutively expresses activeStat3. Preferably, this is carried out as a screening step prior to useof the compound. For example, in the treatment method, thisdetermination can be made by measuring a level of Stat3 activity orinteraction in a biological sample collected from the subject andcomparing the measured level to a reference level of Stat3 activity orinteraction. The biological sample will be appropriate and informativefor the disorder or condition in question. For example, if the disorderor condition is a cancer, a sample of one or more of the cancer cellsshould be collected from the subject. The sample may be a bodily fluidor tissue sample, for example. In the apoptosis induction method, thisdetermination can be made by measuring a level of Stat3 activity orinteraction in the cell to be contacted or in another cell that isrepresentative of the cell to be contacted with the compound (e.g., acell in proximity to the target cell or a cell of the same type orapparently suffering from the same disorder or condition as the targetcell). For example, if the cell is a tumor cell, one or more samples ofthe tumor may be taken to determine Stat3 status. Preferably, thedetermination is made prior to administering the compound to the subjector contacting the cell with the compound. As used herein, singular termssuch as “cell” are inclusive of the plural form such as “cells”. Forexample, the compound may be administered to a single cell or aplurality of cells. In some embodiments, the plurality of cells may be atissue.

Methods for directly or indirectly measuring the level of Stat activityor interaction (such as Stat3 activity or interaction) are known in theart (see, for example, Turkson et al., 1998; Bromberg et al., 1998;Garcia et al., 1997; Nielsen et al., 1997; Catlett-Falcone et al., 1999;Nielsen et al., 1999; Bromberg, 2000; Grandis et al., 2000; Garcia etal., 2001; Epling-Burnette et al., 2001; Epling-Burnette et al., 2001;Bowman et al., 2000b; Niu et al., 2002; Wang et al., 2004a, which areeach incorporated herein by reference in its entirety). Various Stat3measurement methods known in the art and/or disclosed herein may be usedto assess the Stat3 status of a cell or disorder/condition.

The subject invention also concerns a packaged dosage formulationcomprising in one or more containers an inhibitor compound orcomposition of the invention. In one embodiment, a packaged dosageformulation comprises a compound designated herein as PLATINUM-401(formula I). In another embodiment, the compound is a chloroplatinicacid (H₂PtCl₆ or K₂PtCl₆). In still a further embodiment, the compoundis the compound designated herein as RPM 1581 which can be in a freebase (formula II) or salt form (e.g., formula III). A packaged dosageformulation can optionally comprise in one or more containers apharmaceutically acceptable carrier or diluent. A packaged dosageformulation can also optionally comprise, in addition to an inhibitorcompound or composition of the invention, other Stat3 inhibitors,including, but not limited to, NSC 74859, PIAS3, WP1066, or STA-21.

In vivo application of the subject compound, and compositions containingit, can be accomplished by any suitable method and technique presentlyor prospectively known to those skilled in the art. The subject compoundcan be formulated in a physiologically- or pharmaceutically-acceptableform and administered by any suitable route known in the art including,for example, oral, nasal, rectal, and parenteral routes ofadministration. As used herein, the term parenteral includessubcutaneous, intradermal, intravenous, intramuscular, intraperitoneal,and intrasternal administration, such as by injection. Administration ofthe subject compound of the invention can be a single administration, orat continuous or distinct intervals as can be readily determined by aperson skilled in the art.

The compound of the subject invention, and compositions comprising it,can also be administered utilizing liposome technology, slow releasecapsules, implantable pumps, and biodegradable containers. Thesedelivery methods can, advantageously, provide a uniform dosage over anextended period of time. The compounds of the invention can also beadministered in their salt derivative forms or crystalline forms.

Compounds of the subject invention can be formulated according to knownmethods for preparing physiologically acceptable compositions.Formulations are described in detail in a number of sources which arewell known and readily available to those skilled in the art. Forexample, Remington's Pharmaceutical Science by E. W. Martin describesformulations which can be used in connection with the subject invention.In general, the compositions of the subject invention will be formulatedsuch that an effective amount of the compound is combined with asuitable carrier in order to facilitate effective administration of thecomposition. The compositions used in the present methods can also be ina variety of forms. These include, for example, solid, semi-solid, andliquid dosage forms, such as tablets, pills, powders, liquid solutionsor suspension, suppositories, injectable and infusible solutions, andsprays. The preferred form depends on the intended mode ofadministration and therapeutic application. The compositions alsopreferably include conventional physiologically-acceptable carriers anddiluents which are known to those skilled in the art. Examples ofcarriers or diluents for use with the subject compounds include ethanol,dimethyl sulfoxide, glycerol, alumina, starch, saline, and equivalentcarriers and diluents. To provide for the administration of such dosagesfor the desired therapeutic treatment, compositions of the inventionwill advantageously comprise between about 0.1% and 99%, and especially,1 and 15% by weight of the total of one or more of the subject compoundsbased on the weight of the total composition including carrier ordiluent.

Compounds of the invention, and compositions comprising them, can bedelivered to a cell either through direct contact with the cell or via acarrier means. Carrier means for delivering compounds and compositionsto cells are known in the art and include, for example, encapsulatingthe composition in a liposome moiety. Another means for delivery ofcompounds and compositions of the invention to a cell comprisesattaching the compounds to a protein or nucleic acid that is targetedfor delivery to the target cell. U.S. Pat. No. 6,960,648 and PublishedU.S. Patent Application Nos. 20030032594 and 20020120100 disclose aminoacid sequences that can be coupled to another composition and thatallows the composition to be translocated across biological membranes.Published U.S. Patent Application No. 20020035243 also describescompositions for transporting biological moieties across cell membranesfor intracellular delivery. Compounds can also be incorporated intopolymers, examples of which include poly(D-L lactide-co-glycolide)polymer for intracranial tumors; poly[bis(p-carboxyphenoxy)propane:sebacic acid] in a 20:80 molar ratio (as used in GLIADEL);chondroitin; chitin; and chitosan.

The subject invention also concerns methods for treating oncologicaldisorders in a patient. In one embodiment, an effective amount of one ormore compounds or compositions of the present invention is administeredto a patient having an oncological disorder and who is in need oftreatment thereof. Methods of the invention can optionally includeidentifying a patient who is or may be in need of treatment of anoncological disorder. The patient can be a human or other mammal, suchas a primate (monkey, chimpanzee, ape, etc.), dog, cat, cow, pig, orhorse, or other animals having an oncological disorder. Means foradministering and formulating compounds for administration to a patientare known in the art, examples of which are described herein.Oncological disorders within the scope of the invention include, but arenot limited to, cancer and/or tumors of the anus, bile duct, bladder,bone, bone marrow, bowel (including colon and rectum), breast, eye, gallbladder, kidney, mouth, larynx, esophagus, stomach, testis, cervix,head, neck, ovary, lung, mesothelioma, neuroendocrine, penis, skin,spinal cord, thyroid, vagina, vulva, uterus, liver, muscle, pancreas,prostate, blood cells (including lymphocytes and other immune systemcells), and brain. Specific cancers contemplated for treatment with thepresent invention include prostate cancer, head and neck squamous cellcarcinoma, lymphomas, leukemias, and breast cancer.

Examples of cancers that can be treated according to the presentinvention are listed in Table 1.

TABLE 1 Examples of Cancer Types Acute Lymphoblastic Leukemia, AdultHairy Cell Leukemia Acute Lymphoblastic Leukemia, Head and Neck CancerChildhood Hepatocellular (Liver) Cancer, Adult Acute Myeloid Leukemia,Adult (Primary) Acute Myeloid Leukemia, Childhood Hepatocellular (Liver)Cancer, Childhood Adrenocortical Carcinoma (Primary) AdrenocorticalCarcinoma, Childhood Hodgkin's Lymphoma, Adult AIDS-Related CancersHodgkin's Lymphoma, Childhood AIDS-Related Lymphoma Hodgkin's LymphomaDuring Pregnancy Anal Cancer Hypopharyngeal Cancer Astrocytoma,Childhood Cerebellar Hypothalamic and Visual Pathway Glioma,Astrocytoma, Childhood Cerebral Childhood Basal Cell CarcinomaIntraocular Melanoma Bile Duct Cancer, Extrahepatic Islet Cell Carcinoma(Endocrine Pancreas) Bladder Cancer Kaposi's Sarcoma Bladder Cancer,Childhood Kidney (Renal Cell) Cancer Bone Cancer, Osteosarcoma/MalignantKidney Cancer, Childhood Fibrous Histiocytoma Laryngeal Cancer BrainStem Glioma, Childhood Laryngeal Cancer, Childhood Brain Tumor, AdultLeukemia, Acute Lymphoblastic, Adult Brain Tumor, Brain Stem Glioma,Leukemia, Acute Lymphoblastic, Childhood Childhood Leukemia, AcuteMyeloid, Adult Brain Tumor, Cerebellar Astrocytoma, Leukemia, AcuteMyeloid, Childhood Childhood Leukemia, Chronic Lymphocytic Brain Tumor,Cerebral Leukemia, Chronic Myelogenous Astrocytoma/Malignant Glioma,Leukemia, Hairy Cell Childhood Lip and Oral Cavity Cancer Brain Tumor,Ependymoma, Childhood Liver Cancer, Adult (Primary) Brain Tumor,Medulloblastoma, Liver Cancer, Childhood (Primary) Childhood LungCancer, Non-Small Cell Brain Tumor, Supratentorial Primitive LungCancer, Small Cell Neuroectodermal Tumors, Childhood Lymphoma,AIDS-Related Brain Tumor, Visual Pathway and Lymphoma, Burkitt'sHypothalamic Glioma, Childhood Lymphoma, Cutaneous T-Cell, see MycosisBrain Tumor, Childhood Fungoides and Sézary Syndrome Breast CancerLymphoma, Hodgkin's, Adult Breast Cancer, Childhood Lymphoma, Hodgkin's,Childhood Breast Cancer, Male Lymphoma, Hodgkin's During PregnancyBronchial Adenomas/Carcinoids, Lymphoma, Non-Hodgkin's, Adult ChildhoodLymphoma, Non-Hodgkin's, Childhood Burkitt's Lymphoma Lymphoma,Non-Hodgkin's During Carcinoid Tumor, Childhood Pregnancy CarcinoidTumor, Gastrointestinal Lymphoma, Primary Central Nervous SystemCarcinoma of Unknown Primary Macroglobulinemia, Waldenström's CentralNervous System Lymphoma, Malignant Fibrous Histiocytoma of PrimaryBone/Osteosarcoma Cerebellar Astrocytoma, Childhood Medulloblastoma,Childhood Cerebral Astrocytoma/Malignant Melanoma Glioma, ChildhoodMelanoma, Intraocular (Eye) Cervical Cancer Merkel Cell CarcinomaChildhood Cancers Mesothelioma, Adult Malignant Chronic LymphocyticLeukemia Mesothelioma, Childhood Chronic Myelogenous Leukemia MetastaticSquamous Neck Cancer with Chronic Myeloproliferative Disorders OccultPrimary Colon Cancer Multiple Endocrine Neoplasia Syndrome, ColorectalCancer, Childhood Childhood Cutaneous T-Cell Lymphoma, see MultipleMyeloma/Plasma Cell Neoplasm Mycosis Fungoides and Sézary MycosisFungoides Syndrome Myelodysplastic Syndromes Endometrial CancerMyelodysplastic/Myeloproliferative Diseases Ependymoma, ChildhoodMyelogenous Leukemia, Chronic Esophageal Cancer Myeloid Leukemia, AdultAcute Esophageal Cancer, Childhood Myeloid Leukemia, Childhood AcuteEwing's Family of Tumors Myeloma, Multiple Extracranial Germ Cell Tumor,Myeloproliferative Disorders, Chronic Childhood Nasal Cavity andParanasal Sinus Cancer Extragonadal Germ Cell Tumor NasopharyngealCancer Extrahepatic Bile Duct Cancer Nasopharyngeal Cancer, ChildhoodEye Cancer, Intraocular Melanoma Neuroblastoma Eye Cancer,Retinoblastoma Non-Hodgkin's Lymphoma, Adult Gallbladder CancerNon-Hodgkin's Lymphoma, Childhood Gastric (Stomach) Cancer Non-Hodgkin'sLymphoma During Pregnancy Gastric (Stomach) Cancer, Childhood Non-SmallCell Lung Cancer Gastrointestinal Carcinoid Tumor Oral Cancer, ChildhoodGerm Cell Tumor, Extracranial, Oral Cavity Cancer, Lip and ChildhoodOropharyngeal Cancer Germ Cell Tumor, ExtragonadalOsteosarcoma/Malignant Fibrous Germ Cell Tumor, Ovarian Histiocytoma ofBone Gestational Trophoblastic Tumor Ovarian Cancer, Childhood Glioma,Adult Ovarian Epithelial Cancer Glioma, Childhood Brain Stem OvarianGerm Cell Tumor Glioma, Childhood Cerebral Ovarian Low MalignantPotential Tumor Astrocytoma Pancreatic Cancer Glioma, Childhood VisualPathway and Pancreatic Cancer, Childhood Hypothalamic Pancreatic Cancer,Islet Cell Skin Cancer (Melanoma) Paranasal Sinus and Nasal CavityCancer Skin Carcinoma, Merkel Cell Parathyroid Cancer Small Cell LungCancer Penile Cancer Small Intestine Cancer Pheochromocytoma Soft TissueSarcoma, Adult Pineoblastoma and Supratentorial Primitive Soft TissueSarcoma, Childhood Neuroectodermal Tumors, Childhood Squamous CellCarcinoma, see Skin Pituitary Tumor Cancer (non-Melanoma) Plasma CellNeoplasm/Multiple Myeloma Squamous Neck Cancer with OccultPleuropulmonary Blastoma Primary, Metastatic Pregnancy and Breast CancerStomach (Gastric) Cancer Pregnancy and Hodgkin's Lymphoma Stomach(Gastric) Cancer, Childhood Pregnancy and Non-Hodgkin's LymphomaSupratentorial Primitive Primary Central Nervous System LymphomaNeuroectodermal Tumors, Childhood Prostate Cancer T-Cell Lymphoma,Cutaneous, see Rectal Cancer Mycosis Fungoides and Sézary Renal Cell(Kidney) Cancer Syndrome Renal Cell (Kidney) Cancer, ChildhoodTesticular Cancer Renal Pelvis and Ureter, Transitional Cell Thymoma,Childhood Cancer Thymoma and Thymic Carcinoma Retinoblastoma ThyroidCancer Rhabdomyosarcoma, Childhood Thyroid Cancer, Childhood SalivaryGland Cancer Transitional Cell Cancer of the Renal Salivary GlandCancer, Childhood Pelvis and Ureter Sarcoma, Ewing's Family of TumorsTrophoblastic Tumor, Gestational Sarcoma, Kaposi's Unknown Primary Site,Carcinoma of, Sarcoma, Soft Tissue, Adult Adult Sarcoma, Soft Tissue,Childhood Unknown Primary Site, Cancer of, Sarcoma, Uterine ChildhoodSezary Syndrome Unusual Cancers of Childhood Skin Cancer (non-Melanoma)Ureter and Renal Pelvis, Transitional Skin Cancer, Childhood Cell CancerUrethral Cancer Uterine Cancer, Endometrial Uterine Sarcoma VaginalCancer Visual Pathway and Hypothalamic Glioma, Childhood Vulvar CancerWaldenström's Macroglobulinemia Wilms' Tumor

As used herein, the term “tumor” refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues. For example, a particular cancer may becharacterized by a solid mass tumor. The solid tumor mass, if present,may be a primary tumor mass. A primary tumor mass refers to a growth ofcancer cells in a tissue resulting from the transformation of a normalcell of that tissue. In most cases, the primary tumor mass is identifiedby the presence of a cyst, which can be found through visual orpalpation methods, or by irregularity in shape, texture or weight of thetissue. However, some primary tumors are not palpable and can bedetected only through medical imaging techniques such as X-rays (e.g.,mammography), or by needle aspirations. The use of these lattertechniques is more common in early detection. Molecular and phenotypicanalysis of cancer cells within a tissue will usually confirm if thecancer is endogenous to the tissue or if the lesion is due to metastasisfrom another site.

For the treatment of oncological disorders, the compounds andcompositions of this invention can be administered to a patient in needof treatment in combination with other antitumor or anticancersubstances and/or with radiation and/or photodynamic therapy and/or withsurgical treatment to remove a tumor. These other substances ortreatments may be given at the same as or at different times from thecompounds and compositions of this invention. For example, the compoundsof the present invention can be used in combination with mitoticinhibitors such as taxol or vinblastine, alkylating agents such ascyclophosamide or ifosfamide, antimetabolites such as 5-fluorouracil orhydroxyurea, DNA intercalators such as adriamycin or bleomycin,topoisomerase inhibitors such as etoposide or camptothecin,antiangiogenic agents such as angiostatin, antiestrogens such astamoxifen, and/or other anti-cancer drugs or antibodies, such as, forexample, GLEEVEC (Novartis Pharmaceuticals Corporation) and HERCEPTIN(Genentech, Inc.), respectively. In one embodiment, compounds andcompositions of the invention can be used in combination with otherStat3 inhibitors, including, but not limited to, NSC 74859, PIAS3,WP1066, or STA-21.

The methods of the present invention can be used with humans and otheranimals. As used herein, the terms “subject”, “patient”, and“individual” refer to a human or non-human animal. The other animalscontemplated within the scope of the invention include domesticated,agricultural, or zoo- or circus-maintained animals. Domesticated animalsinclude, for example, dogs, cats, rabbits, ferrets, guinea pigs,hamsters, pigs, monkeys or other primates, and gerbils. Agriculturalanimals include, for example, horses, mules, donkeys, burros, cattle,cows, pigs, sheep, and alligators. Zoo- or circus-maintained animalsinclude, for example, lions, tigers, bears, camels, giraffes,hippopotamuses, and rhinoceroses. In one embodiment, the subject is amammal. In one embodiment, the mammalian subject is a human. In anotherembodiment, the mammalian subject is a non-human mammal.

While inhibitor compounds of the invention can be administered to asubject or contacted with a cell as isolated compounds, these compoundscan also be administered or contacted as part of a pharmaceuticalcomposition. The subject invention thus further provides compositionscomprising one or more compounds in association with at least onepharmaceutically acceptable carrier. The pharmaceutical composition canbe adapted for various routes of administration, such as enteral,parenteral, intravenous, intramuscular, topical, subcutaneous, and soforth. Administration or contact can be continuous or at distinctintervals, as can be determined by a person of ordinary skill in theart. In cases where the cells are contacted in vivo, the cells arecontacted by administration of the compound to a subject with the cells.The compound may be administered to a subject locally at a desired siteof action or systemically.

The inhibitor compounds of the invention can be formulated according toknown methods for preparing pharmaceutically useful compositions.Formulations are described in a number of sources which are well knownand readily available to those skilled in the art. For example,Remington's Pharmaceutical Science (Martin 1995) describes formulationswhich can be used in connection with the subject invention. Formulationssuitable for administration include, for example, aqueous sterileinjection solutions, which may contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient; and aqueous and nonaqueous sterilesuspensions which may include suspending agents and thickening agents.The formulations may be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in a freezedried (lyophilized) condition requiring only the condition of thesterile liquid carrier, for example, water for injections, prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powder, granules, tablets, etc. It should be understood that inaddition to the ingredients particularly mentioned above, thecompositions of the subject invention can include other agentsconventional in the art having regard to the type of formulation inquestion.

Therapeutic application of compounds and/or compositions containing themcan be accomplished by any suitable therapeutic method and techniquepresently or prospectively known to those skilled in the art. Further,compounds and agents of the invention have use as starting materials orintermediates for the preparation of other useful compounds andcompositions.

Compounds of the invention, and compositions thereof, may be locallyadministered at one or more anatomical sites, such as sites of unwantedcell growth (such as a tumor site or benign skin growth, e.g., injectedor topically applied to the tumor or skin growth) or sites of fungalinfection, optionally in combination with a pharmaceutically acceptablecarrier such as an inert diluent. Compounds of the invention, andcompositions thereof, may be systemically administered, such asintravenously or orally, optionally in combination with apharmaceutically acceptable carrier such as an inert diluent, or anassimilable edible carrier for oral delivery. They may be enclosed inhard or soft shell gelatin capsules, may be compressed into tablets, ormay be incorporated directly with the food of the patient's diet. Fororal therapeutic administration, the active compound may be combinedwith one or more excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,aerosol sprays, and the like.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac, or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

Compounds and compositions of the invention, including pharmaceuticallyacceptable salts or analogs thereof, can be administered intravenously,intramuscularly, or intraperitoneally by infusion or injection.Solutions of the active agent or its salts can be prepared in water,optionally mixed with a nontoxic surfactant. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, triacetin, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations can contain a preservative to prevent the growthof microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. The ultimatedosage form should be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium comprising, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants. Optionally, the prevention of the action of microorganismscan be brought about by various other antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the inclusion of agents that delay absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating a compound ofthe invention in the required amount in the appropriate solvent withvarious other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

For topical administration, compounds of the invention may be applied inas a liquid or solid. However, it will generally be desirable toadminister them topically to the skin as compositions, in combinationwith a dermatologically acceptable carrier, which may be a solid or aliquid. Compounds and compositions of the subject invention can beapplied topically to a subject's skin to reduce the size (and mayinclude complete removal) of malignant or benign growths, or to treat aninfection site. Compounds of the invention can be applied directly tothe growth or infection site. Preferably, the compounds and agents areapplied to the growth or infection site in a formulation such as anointment, cream, lotion, solution, tincture, or the like. Drug deliverysystems for delivery of pharmacological substances to dermal lesions canalso be used, such as that described in U.S. Pat. No. 5,167,649.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers, for example.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user. Examples of useful dermatological compositionswhich can be used to deliver a compound to the skin are disclosed inU.S. Pat. Nos. 4,608,392; 4,992,478; 4,559,157; and 4,820,508.

Useful dosages of the compounds and pharmaceutical compositions of thepresent invention can be determined by comparing their in vitroactivity, and in vivo activity in animal models. Methods for theextrapolation of effective dosages in mice, and other animals, to humansare known to the art; for example, see U.S. Pat. No. 4,938,949.

The present invention also concerns pharmaceutical compositionscomprising a compound of the invention in combination with apharmaceutically acceptable carrier. Pharmaceutical compositions adaptedfor oral, topical or parenteral administration, comprising an amount ofa compound constitute a preferred embodiment of the invention. The doseadministered to a patient, particularly a human, in the context of thepresent invention should be sufficient to achieve a therapeutic responsein the patient over a reasonable time frame, without lethal toxicity,and preferably causing no more than an acceptable level of side effectsor morbidity. One skilled in the art will recognize that dosage willdepend upon a variety of factors including the condition (health) of thesubject, the body weight of the subject, kind of concurrent treatment,if any, frequency of treatment, therapeutic ratio, as well as theseverity and stage of the pathological condition.

For the treatment of oncological disorders, compounds and compositionscontemplated by the present invention can be administered to a patientin need of treatment prior to, subsequent to, or in combination withother antitumor or anticancer agents or substances (e.g.,chemotherapeutic agents, immunotherapeutic agents, radiotherapeuticagents, cytotoxic agents, etc.) and/or with radiation therapy and/orwith surgical treatment to remove a tumor. For example, compounds andcompositions of the present invention can be used in methods of treatingcancer wherein the patient is to be treated or is or has been treatedwith mitotic inhibitors such as taxol or vinblastine, alkylating agentssuch as cyclophosamide or ifosfamide, antimetabolites such as5-fluorouracil or hydroxyurea, DNA intercalators such as adriamycin orbleomycin, topoisomerase inhibitors such as etoposide or camptothecin,antiangiogenic agents such as angiostatin, antiestrogens such astamoxifen, and/or other anti-cancer drugs or antibodies, such as, forexample, GLEEVEC (Novartis Pharmaceuticals Corporation) and HERCEPTIN(Genentech, Inc.), respectively. These other substances or radiationtreatments may be given at the same as or at different times from thecompounds of this invention. Examples of other chemotherapeutic agentscontemplated within the scope of the invention include, but are notlimited to, altretamine, bleomycin, bortezomib (VELCADE), busulphan,calcium folinate, capecitabine, carboplatin, carmustine, chlorambucil,cisplatin, cladribine, crisantaspase, cyclophosphamide, cytarabine,dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin,epirubicin, etoposide, fludarabine, fluorouracil, gefitinib (IRESSA),gemcitabine, hydroxyurea, idarubicin, ifosfamide, imatinib (GLEEVEC),irinotecan, liposomal doxorubicin, lomustine, melphalan, mercaptopurine,methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel,pentostatin, procarbazine, raltitrexed, streptozocin, tegafur-uracil,temozolomide, thiotepa, tioguanine/thioguanine, topotecan, treosulfan,vinblastine, vincristine, vindesine, vinorelbine. In an exemplifiedembodiment, the chemotherapeutic agent is melphalan. Examples ofimmunotherapeutic agents contemplated within the scope of the inventioninclude, but are not limited to, alemtuzumab, cetuximab (ERBITUX),gemtuzumab, iodine 131 tositumomab, rituximab, trastuzamab (HERCEPTIN).Cytotoxic agents include, for example, radioactive isotopes (e.g., I¹³¹,I¹²⁵, Y⁹⁰, P³², etc.), and toxins of bacterial, fungal, plant, or animalorigin (e.g., ricin, botulinum toxin, anthrax toxin, aflatoxin,jellyfish venoms (e.g., box jellyfish), etc.) The subject invention alsoconcerns methods for treating an oncological disorder comprisingadministering an effective amount of a compound and/or composition ofthe invention prior to, subsequent to, and/or in combination withadministration of a chemotherapeutic agent, an immunotherapeutic agent,a radiotherapeutic agent, or radiotherapy.

Examples of some chemotherapeutic agents that can be used according tothe present invention are listed in Table 2.

TABLE 2 Examples of Chemotherapeutic Agents 13-cis-Retinoic Acid Mylocel2-Amino-6- Letrozole Mercaptopurine Neosar 2-CdA Neulasta2-Chlorodeoxyadenosine Neumega 5-fluorouracil Neupogen 5-FU Nilandron6-TG Nilutamide 6-Thioguanine Nitrogen Mustard 6-Mercaptopurine Novaldex6-MP Novantrone Accutane Octreotide Actinomycin-D Octreotide acetateAdriamycin Oncospar Adrucil Oncovin Agrylin Ontak Ala-Cort OnxalAldesleukin Oprevelkin Alemtuzumab Orapred Alitretinoin OrasoneAlkaban-AQ Oxaliplatin Alkeran Paclitaxel All-transretinoic acidPamidronate Alpha interferon Panretin Altretamine ParaplatinAmethopterin Pediapred Amifostine PEG Interferon AminoglutethimidePegaspargase Anagrelide Pegfilgrastim Anandron PEG-INTRON AnastrozolePEG-L-asparaginase Arabinosylcytosine Phenylalanine Mustard Ara-CPlatinol Aranesp Platinol-AQ Aredia Prednisolone Arimidex PrednisoneAromasin Prelone Arsenic trioxide Procarbazine Asparaginase PROCRIT ATRAProleukin Avastin Prolifeprospan 20 with Carmustine implant BCGPurinethol BCNU Raloxifene Bevacizumab Rheumatrex Bexarotene RituxanBicalutamide Rituximab BiCNU Roveron-A (interferon alfa-2a) BlenoxaneRubex Bleomycin Rubidomycin hydrochloride Bortezomib SandostatinBusulfan Sandostatin LAR Busulfex Sargramostim C225 Solu-Cortef CalciumLeucovorin Solu-Medrol Campath STI-571 Camptosar StreptozocinCamptothecin-11 Tamoxifen Capecitabine Targretin Carac Taxol CarboplatinTaxotere Carmustine Temodar Carmustine wafer Temozolomide CasodexTeniposide CCNU TESPA CDDP Thalidomide CeeNU Thalomid CerubidineTheraCys cetuximab Thioguanine Chlorambucil Thioguanine TabloidCisplatin Thiophosphoamide Citrovorum Factor Thioplex CladribineThiotepa Cortisone TICE Cosmegen Toposar CPT-11 TopotecanCyclophosphamide Toremifene Cytadren Trastuzumab Cytarabine TretinoinCytarabine liposomal Trexall Cytosar-U Trisenox Cytoxan TSPA DacarbazineVCR Dactinomycin Velban Darbepoetin alfa Velcade Daunomycin VePesidDaunorubicin Vesanoid Daunorubicin Viadur hydrochloride VinblastineDaunorubicin liposomal Vinblastine Sulfate DaunoXome Vincasar PfsDecadron Vincristine Delta-Cortef Vinorelbine Deltasone Vinorelbinetartrate Denileukin diftitox VLB DepoCyt VP-16 Dexamethasone VumonDexamethasone acetate Xeloda dexamethasone sodium Zanosar phosphateZevalin Dexasone Zinecard Dexrazoxane Zoladex DHAD Zoledronic acid DICZometa Diodex Gliadel wafer Docetaxel Glivec Doxil GM-CSF DoxorubicinGoserelin Doxorubicin liposomal granulocyte - colony stimulating factorDroxia Granulocyte macrophage colony stimulating DTIC factor DTIC-DomeHalotestin Duralone Herceptin Efudex Hexadrol Eligard Hexalen EllenceHexamethylmelamine Eloxatin HMM Elspar Hycamtin Emcyt Hydrea EpirubicinHydrocort Acetate Epoetin alfa Hydrocortisone Erbitux Hydrocortisonesodium phosphate Erwinia L-asparaginase Hydrocortisone sodium succinateEstramustine Hydrocortone phosphate Ethyol Hydroxyurea EtopophosIbritumomab Etoposide Ibritumomab Tiuxetan Etoposide phosphate IdamycinEulexin Idarubicin Evista Ifex Exemestane IFN-alpha Fareston IfosfamideFaslodex IL-2 Femara IL-11 Filgrastim Imatinib mesylate FloxuridineImidazole Carboxamide Fludara Interferon alfa Fludarabine InterferonAlfa-2b (PEG conjugate) Fluoroplex Interleukin-2 FluorouracilInterleukin-11 Fluorouracil (cream) Intron A (interferon alfa-2b)Fluoxymesterone Leucovorin Flutamide Leukeran Folinic Acid Leukine FUDRLeuprolide Fulvestrant Leurocristine G-CSF Leustatin Gefitinib LiposomalAra-C Gemcitabine Liquid Pred Gemtuzumab ozogamicin Lomustine GemzarL-PAM Gleevec L-Sarcolysin Lupron Meticorten Lupron Depot MitomycinMatulane Mitomycin-C Maxidex Mitoxantrone Mechlorethamine M-PrednisolMechlorethamine MTC Hydrochlorine MTX Medralone Mustargen Medrol MustineMegace Mutamycin Megestrol Myleran Megestrol Acetate Iressa MelphalanIrinotecan Mercaptopurine Isotretinoin Mesna Kidrolase Mesnex LanacortMethotrexate L-asparaginase Methotrexate Sodium LCR Methylprednisolone

The subject invention also concerns methods for inhibiting Stat3 proteinin a cell by contacting the cell with an effective amount of a compoundor composition of the invention. In one embodiment, the cell is a humanor mammalian cell, and can be a cancer or tumor cell or other cell thatexhibits abnormal proliferation, survival, migration or differentiation.In one embodiment, the cell constitutively expresses or expresseselevated or abnormal levels of Stat3. In one embodiment, the tumor orcancer cell is a breast cancer cell, prostate cancer cell, head and necksquamous cell carcinoma, lymphoma cell, leukemia cell, multiple myelomacell, glioma cell, non-small cell lung cancer cell, melanoma cell,gastrointestinal stromal tumor cell, renal cell carcinoma, esophagealcell carcinoma, ovarian cancer cell, cervical cancer cell, or gastriccancer cell. In one embodiment, the compound is the compound designatedherein as PLATINUM-401 (formula I). In another embodiment, the compoundis a chloroplatinic acid (H₂PtCl₆ or K₂PtCl₆). In still a furtherembodiment, the compound is the compound designated herein as RPM 1581which can be in a free base (formula II) or salt form (e.g., formulaIII).

The subject invention also concerns methods for treating a person oranimal (i.e., a subject) having a disorder associated with constitutive,abnormal, or elevated expression of Stat3 in a cell, wherein atherapeutically effective amount of a compound or composition of theinvention is administered to the person or animal. The disorder can beone characterized, for example, by abnormal cell proliferation, cellsurvival, cell migration, and/or cell differentiation. In oneembodiment, the compound is the compound designated herein asPLATINUM-401 (formula I). In another embodiment, the compound is achloroplatinic acid (H₂PtCl₆ or K₂PtCl₆). In still a further embodiment,the compound is the compound designated herein as RPM 1581 which can bein a free base (formula II) or salt form (e.g., formula III).

Depending upon the disorder or disease condition to be treated, asuitable dose(s) may be that amount that will reduce proliferation orgrowth of the target cell(s). In the context of cancer, a suitabledose(s) is that which will result in a concentration of the active agentin cancer tissue, such as a malignant tumor, which is known to achievethe desired response. The preferred dosage is the amount which resultsin maximum inhibition of cancer cell growth, without unmanageable sideeffects. Administration of a compound and/or agent can be continuous orat distinct intervals, as can be determined by a person of ordinaryskill in the art.

To provide for the administration of such dosages for the desiredtherapeutic treatment, in some embodiments, pharmaceutical compositionsof the invention can comprise between about 0.1% and 45%, andespecially, 1 and 15%, by weight of the total of one or more of thecompounds based on the weight of the total composition including carrieror diluents. Illustratively, dosage levels of the administered activeingredients can be: intravenous, 0.01 to about 20 mg/kg;intraperitoneal, 0.01 to about 100 mg/kg; subcutaneous, 0.01 to about100 mg/kg; intramuscular, 0.01 to about 100 mg/kg; orally 0.01 to about200 mg/kg, and preferably about 1 to 100 mg/kg; intranasal instillation,0.01 to about 20 mg/kg; and aerosol, 0.01 to about 20 mg/kg of animal(body) weight.

The subject invention also concerns methods for screening and/ordiagnosing an oncological condition or disorder using a compound orcomposition of the invention wherein the condition or disorder isassociated with constitutive active Stat3 expression or active Stat3overexpression in a cell. In one embodiment, a cell is contacted with aPLATINUM-401 compound or composition and observing the cell forinduction of apoptosis. Only cells expressing constitutively activeStat3 will exhibit induction of apoptosis upon exposure to PLATINUM-401(formula I). In another embodiment, the compound is a chloroplatinicacid (H₂PtCl₆ or K₂PtCl₆). In still a further embodiment, the compoundis the compound designated herein as RPM 1581 which can be in a freebase (formula II) or salt form (e.g., formula III).

The subject invention also concerns kits comprising a compound or acomposition comprising an inhibitor compound and/or agent of theinvention in one or more containers. In one embodiment, the compound isthe compound designated herein as PLATINUM-401 (formula I). In anotherembodiment, the compound is a chloroplatinic acid (H₂PtCl₆ or K₂PtCl₆).In still a further embodiment, the compound is the compound designatedherein as RPM 1581 which can be in a free base (formula II) or salt form(e.g., formula III). Kits of the invention can optionally includepharmaceutically acceptable carriers and/or diluents. In one embodiment,a kit of the invention includes one or more other components, adjuncts,or adjuvants as described herein. In another embodiment, a kit includesone or more anti-cancer agents, such as those agents described herein.In one embodiment, a kit of the invention includes instructions orpackaging materials that describe how to administer a compound orcomposition of the kit. Containers of the kit can be of any suitablematerial, e.g., glass, plastic, metal, etc., and of any suitable size,shape, or configuration. In one embodiment, a compound and/or agent ofthe invention is provided in the kit as a solid, such as a tablet, pill,or powder form. In another embodiment, a compound and/or agent of theinvention is provided in the kit as a liquid or solution. In oneembodiment, the kit comprises an ampoule or syringe containing acompound and/or agent of the invention in liquid or solution form. A kitof the invention can also optionally comprise, in addition to aninhibitor compound or composition of the invention, other Stat3inhibitors, including, but not limited to, NSC 74859, PIAS3, WP1066, orSTA-21.

Mammalian species which benefit from the disclosed methods include, butare not limited to, primates, such as apes, chimpanzees, orangutans,humans, monkeys; domesticated animals (e.g., pets) such as dogs, cats,guinea pigs, hamsters, Vietnamese pot-bellied pigs, rabbits, andferrets; domesticated farm animals such as cows, buffalo, bison, horses,donkey, swine, sheep, and goats; exotic animals typically found in zoos,such as bear, lions, tigers, panthers, elephants, hippopotamus,rhinoceros, giraffes, antelopes, sloth, gazelles, zebras, wildebeests,prairie dogs, koala bears, kangaroo, opossums, raccoons, pandas, hyena,seals, sea lions, elephant seals, otters, porpoises, dolphins, andwhales. Other species that may benefit from the disclosed methodsinclude fish, amphibians, avians, and reptiles. As used herein, theterms “patient”, “individual”, and “subject” are used interchangeablyand are intended to include such human and non-human species. Likewise,in vitro methods of the present invention can be carried out on cells ofsuch human and non-human species.

The compound designated herein as PLATINUM-401 functions as a Stat3inhibitor by directly interacting with the protein. The evidenceindicates that PLATINUM-401 interacts with the Stat3 protein, both theinactive monomer and the activated dimer, and represses the Stat3phosphotyrosine levels, DNA-binding activity and transcriptionalregulation. Differences are evident in the modes of activity andselectivity between PLATINUM-401 and the widely used anti-tumor agentCisplatin (Siddik, 2003; Wang et al., 2004b), which has no effect onStat3 activity (Turkson et al., 2004b). By contrast, PLATINUM-401 blocksthe binding of activated Stat3 to a specific DNA-response element. Whilethe exact site(s) within the Stat3 protein that interacts withPLATINUM-401 is not yet known, preliminary evidence (data not shown)implicates cysteine residue(s). This is consistent with previous reportsthat Cisplatin and other platinum complexes interact with cysteine andmethionine residues in serum albumin and γ-globulins (Bose, 2002;Trynda-Lemiesz et al., 1999; Allain et al., 2000; Trynda-Lemiesz andLuczkowski, 2004), forming thiol conjugates of platinum complexes(Allain et al., 2000; Heudi et al., 1999; Heudi et al., 2001). It isconceivable that such a modification in the Stat3 protein byPLATINUM-401 in turn occludes the binding of Stat3 to its consensusDNA-response element.

A non-competitive type inhibition by PLATINUM-401 was observed,supported by the reduced maximum DNA-binding activity of Stat3 followingPLATINUM-401 treatment. The treatment of activated Stat3 withPLATINUM-401 also induces an apparent change in the protein's bindingaffinity for the consensus DNA sequence. Inferring from the knowninteractions of other platinum complexes with thiol-containingbiological molecules (Siddik, 2003; Allain et al., 2000; Heudi et al.,1999; Heudi et al., 2001), the expected reaction of PLATINUM-401 withthiol groups of Stat3 would be irreversible. Those earlier studies andours together suggest that PLATINUM-401 irreversibly modifies the Stat3protein, thereby blocking the DNA-binding activity of the protein andsubverting its transcriptional and biological functions. The lack of anyPLATINUM-401 effect on Stat3 protein pre-bound to DNA suggests ashielding of the key amino acid(s) within the protein once the latter isfirst bound to DNA. This raises the possibility that both PLATINUM-401and the DNA consensus sequence bind to the same region of Stat3 in theDNA-binding domain, or the Stat3 protein undergoes conformationalchanges upon binding to the DNA sequence, which restrict access to thekey amino acid residue(s) and prevent interaction with PLATINUM-401.Indeed, the crystal structure of Stat3β dimer bound to DNA (Becker etal., 1998) shows that the protein is clamped around the DNA doublehelix, which may be sufficient to impede access by PLATINUM-401 to theDNA-binding domain.

Previous reports have established constitutively-active Stat3 as key tothe dysregulated growth, survival, angiogenesis, and immune evasion thatcharacterize tumorigenesis (Turkson and Jove, 2000; Yu and Jove, 2004).The biological effects of PLATINUM-401 are manifest in malignant cellsharboring constitutively-active Stat3, including inhibition ofStat3-dependent transformation, as well as inhibition of cell growthwith G₀/G₁ cell cycle arrest and apoptosis of malignant cells(Catlett-Falcone et al. 1999; Garcia et al., 2001; Bowman et al., 2000b;Turkson et al., 2004b). Thus, PLATINUM-401 inhibits Stat3-mediatedinduction of critical genes, including the cell cycle regulator, CyclinD1, the anti-apoptotic Bcl-xL, as well as the pro-angiogenic VEGF, whichare important in tumor processes (Catlett-Falcone et al. 1999; Niu etal., 2002; Bromberg et al., 1999; Sinibaldi et al., 2000).

In contrast to the DNA denaturation and the formation of platinum-DNAadducts by Cisplatin (Siddik, 2003; Perez et al., 2003), directmodification of DNA is not a key factor in the inhibition of Stat3signaling and biological functions by PLATINUM-401. Findings herein showthat the Stat3-binding integrity of the DNA response element ispreserved following treatment of DNA with PLATINUM-401. Moreover,oligonucleotide melting and re-annealing analysis with agarose gelelectrophoresis show a retention of the overall integrity of thePLATINUM-401-treated DNA response element (data not shown). The effectsof PLATINUM-401 on Stat3 observed here also contrast that of Cisplatinand others that modulate the PI-3-kinase/Akt and MAPKs family pathways,which contribute to their biological effects (Sanchez-Perez et al.,1998; Persons et al., 1999; Bose, 2002; Siddik, 2003). The inventors donot observe effects of PLATINUM-401 on Stat3-independent transcriptionalevents.

Exemplified Embodiments:

-   Embodiment 1: A compound having the structure shown in formula I,    formula II, or formula III:

-   Embodiment 2: The compound of embodiment 1, wherein the compound has    the structure of formula I.-   Embodiment 3: The compound of embodiment 1, wherein the compound has    the structure of formula II.-   Embodiment 4: The compound of embodiment 1, wherein the compound has    the structure of formula III.-   Embodiment 5: The compound of embodiment 1, wherein the compound is    a chloroplatinic acid (H₂PtCl₆ or K₂PtCl₆).-   Embodiment 6: The compound of embodiment 1, wherein the compound is    RPM 1581.-   Embodiment 7: A composition comprising a compound of embodiment 1    and a carrier, buffer, adjuvant, a combination of two or more of the    foregoing.-   Embodiment 8: A method for treating a subject having a disorder or    condition associated with aberrant or excessive Stat3 activity or    interaction in a cell, comprising administering an effective amount    of a compound of embodiment 1 to the subject.-   Embodiment 9: The method of embodiment 8, wherein the disorder is an    oncological disorder.-   Embodiment 10: The method of embodiment 8 or 9, wherein the compound    has the structure of formula I.-   Embodiment 11: The method of embodiment 8 or 9, wherein the compound    has the structure of formula II.-   Embodiment 12: The method of embodiment 8 or 9, wherein the compound    has the structure of formula III.-   Embodiment 13: The method of embodiment 8 or 9, wherein the compound    is a chloroplatinic acid (H₂PtCl₆ or K₂PtCl₆).-   Embodiment 14: The method of embodiment 8 or 9, wherein the compound    is RPM 1581.-   Embodiment 15: The method of any preceding embodiment, wherein the    method further comprises, determining that the disorder or condition    is associated with aberrant or excessive Stat3 activity or    interaction in a cell (e.g., prior to administering the compound).-   Embodiment 16: The method of embodiment 15, wherein the determining    comprises measuring a level of Stat3 activity or interaction in a    biological sample collected from the subject and comparing the    measured level to a reference level of Stat3 activity or    interaction.-   Embodiment 17: The method of any preceding embodiment, wherein the    compound is administered to the subject in a composition comprising    the compound and a carrier, buffer, adjuvant, or a combination of    two or more of the foregoing.-   Embodiment 18: The method of any preceding embodiment, wherein the    subject is a mammal.-   Embodiment 19: The method of embodiment 18, wherein the subject is a    non-human mammal.-   Embodiment 20: The method of embodiment 18, wherein the subject is    human.-   Embodiment 21: A method for inducing apoptosis in a cell aberrantly    or constitutively expressing active Stat3, comprising contacting the    cell with an effective amount of a compound of embodiment 1.-   Embodiment 22: The method of embodiment 21, wherein the cell is a    malignant cell.-   Embodiment 23: The method of embodiment 21 or 22, wherein the    compound has the structure of formula I.-   Embodiment 24: The method of embodiment 21 or 22, wherein the    compound has the structure of formula II.-   Embodiment 25: The method of embodiment 21 or 22, wherein the    compound has the structure of formula III.-   Embodiment 26: The method of embodiment 21 or 22, wherein the    compound is a chloroplatinic acid (H₂PtCl₆ or K₂PtCl₆).-   Embodiment 27: The method of embodiment 21 or 22, wherein the    compound is RPM 1581.-   Embodiment 28: The method of any preceding embodiment, wherein the    method further comprises, determining that the cell or a    representative cell aberrantly or constitutively expresses active    Stat3 (e.g., prior to contacting the cell with the compound).-   Embodiment 29: The method of embodiment 28, wherein the determining    comprises measuring a level of Stat3 activity or interaction in the    cell or representative cell and comparing the measured level to a    reference level of Stat3 activity or interaction.-   Embodiment 30: The method of any preceding embodiment, wherein the    contacting comprises contacting the cell with a composition    comprising the compound and a carrier, buffer, adjuvant, or a    combination of two or more of the foregoing.-   Embodiment 31: The method of any preceding embodiment, wherein the    contacting is carried out in vitro.-   Embodiment 32: The method of any one of embodiments 23 to 24,    wherein the contacting is carried out in vivo.-   Embodiment 33: The method of embodiment 30, wherein the contacting    comprises administering the effective amount of the compound to a    human.-   Embodiment 34: A method for screening and/or diagnosing an    oncological condition or disorder using a compound of embodiment 1,    wherein the condition or disorder is associated with constitutive    active Stat3 expression or active Stat3 overexpression in a cell,    wherein the method comprises contacting a cell with a compound of    embodiment 1 and observing the cell for induction of apoptosis.-   Embodiment 35: A kit comprising a compound of embodiment 1 in one or    more containers.

Materials and Methods

Cells and reagents—Src-transformed NIH3T3/v-Src, NIH3T3/v-Src/pLucTKS3,NIH3T3/v-Src/pRLSRE, and NIH3T3/hEGFR fibroblasts, human breast cancer(MDA-MB-231, MDA-MB-435, MDA-MB-453, and MDA-MB-468), human prostatecancer (DU145), multiple myeloma U266 (human) and 5TGM1 (mouse), mousemelanoma (B16), and human pancreatic cancer (Panc1) cell lines have allbeen previously described (Catlett-Falcone et al., 1999; Garcia et al.,2001; Niu et al., 1999; Johnson et al., 1985; Yu et al., 1995; Turksonet al., 2001; Mora et al., 2002; Oyajobi et al., 2003). Cells were grownin Dulbecco's modified Eagle's medium (DMEM) containing 5%iron-supplemented bovine calf serum, with or without G418 or zeocin, orin RPMI containing 10% heat-inactivated fetal bovine serum. Therecombinant human epidermal growth factor (rhEGF) and interleukin-6(IL-6) were obtained from R & D Systems (Minneapolis, Minn.).

Plasmids—The Stat3 reporter, pLucTKS3, Stat3-dependent VEGFpromoter-driven reporter (pGL2-VEGF-Luc), and the Stat3-independentreporter, pLucSRE, all of which drive the expression of fireflyluciferase, as well as the Stat3-independent pRLSRE renilla luciferasereporter have all been previously described (Turkson et al., 1998; Niuet al., 2002; Turkson et al., 1999). The pLucTKS3 plasmid harbors sevencopies of a sequence corresponding to the Stat3-specific binding site inthe promoter of the human C-reactive protein gene (Zhang et al., 1996).The pRLSRE and pLucSRE, each contains two copies of the serum responseelement (SRE) from the c-fos promoter (Turkson et al., 1998; Yamauchi etal., 1993), subcloned into the renilla (pRL-null) or firefly (pGL2)luciferase reporter, respectively (Promega, Madison, Wis.). The plasmidpNFκB-Luc is firefly luciferase obtained from Strategene (La Jolla,Calif.). The plasmid pRc/CMV Stat3 Flag tagged was a gift from Dr. JamesDarnell, Jr. (The Rockefeller University).

Cytosolic extract preparation and luciferase assays—Cytosolic extractpreparation from fibroblasts and luciferase assays were previouslydescribed (Turkson et al., 1998; Turkson et al., 1999). Briefly, aftertwo washes with PBS and equilibration for 5 min with 0.5 ml of PBS-0.5mM EDTA, cells were scraped off the dishes and the cell pellet wasobtained by centrifugation (4,500×g, 2 min, 4° C.). Cells wereresuspended in 0.4 ml of low-salt HEPES buffer (10 mM HEPES [pH 7.8], 10mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride(PMSF), and 1 mM dithiothreitol (DTT)) for 15 min, lysed by the additionof 20 μl of 10% Nonidet P-40 (NP-40), and centrifuged (10,000×g, 30 s,4° C.) to obtain the cytosolic supernatant, which was used forluciferase assays (Promega) measured with a luminometer. Cytosoliclysates were prepared from recombinant baculovirus-infected Sf-9 cellsfor Stat3 protein, as previously described (Zhang et al., 2000).Briefly, cultured dishes of Sf-9 cells were washed twice with ice-cold1×PBS and then PBS containing 1 mM sodium orthovanadate. Cells were thenlysed in 1% NP-40 lysis buffer (50 mM HEPES [pH 7.9], 150 mM NaCl, 1%NP-40, 20 mM NaF, 1 mM sodium orthovanadate, 1 mM tetrasodiumpyrophosphate, 1 mM DTT, 0.5 mM PMSF, 2 mM EGTA, 2 mM EDTA, 0.1 μMaprotinin, 1 μM leupeptin, and 1 μM antipain) on ice for 10 min, andcentrifuged (13,000×g, 30 s, 4° C.) to obtain lysate.

Nuclear extract preparation and gel shift assays—Nuclear extractpreparation from NIH3T3 stimulated by rhIL-6, NIH3T3/hEGFR stimulated byrhEGF, v-Src-transformed fibroblasts (NIH3T3/v-Src) or tumor cell linesand electrophoretic mobility shift assay were carried out as previouslydescribed (Turkson et al., 1998; Garcia et al., 1997; Yu et al., 1995).The ³²P-labeled oligonucleotide probes used are hSIE (high affinitysis-inducible element from the c-fos gene, m67 variant,5′-AGCTTCATTTCCCGTAAATCCCTA) (SEQ ID NO:1) that binds Stat1 and Stat3(Garcia et al., 1997; Wagner et al., 1990) and MGFe (mammary glandfactor element from the bovine β-casein gene promoter,5′-AGATTTCTAGGAATTCAA) (SEQ ID NO:2) for Stat1 and Stat5 binding(49,50). Except where indicated, inhibitor compound was pre-incubatedwith the nuclear extract for 30 min at room temperature prior toincubation with radiolabeled probe.

Western blot—Whole-cell lysates were prepared in boiling sodium dodecylsulfate (SDS) sample-loading buffer to extract total proteins from thecytoplasm and nucleus. Equivalent amounts of total cellular protein wereelectrophoresed on an SDS-10% polyacrylamide gel and transferred ontonitrocellulose membranes. Probing of nitrocellulose membranes withprimary antibodies and detection of horseradish peroxidase-conjugatedsecondary antibodies by enhanced chemiluminescence (Amersham,Piscataway, N.J.) were performed as previously described (Garcia et al.,2001; Turkson et al., 2004b; Zhang et al., 2000). The probes used wereanti-Cyclin D1 (Cell Signaling Technologies, Beverly, Mass.),anti-Bcl-xL (Cell Signaling Technologies), and anti-β-Actin(Sigma-Aldrich, St. Louis, Mo.) antibodies.

Soft-agar colony formation assays—Colony formation assays were carriedout in six-well dishes as previously described (Turkson et al., 1999).In brief, each well contained 1.5 ml of 1% agarose in DMEM as the bottomlayer, and 1.5 ml of 0.5% agarose in DMEM containing 4000 or 6000NIH3T3/v-Src or NIH3T3/v-Ras fibroblasts, respectively, as the toplayer. Treatment with PLATINUM-401 was initiated 1 day after seedingcells by adding 75-100 μl of medium with or without compound, andrepeated every three days, until large colonies were evident. Colonieswere quantified by staining with 20 μl of 1 mg/ml iodonitrotetrazoliumviolet, incubating at 37° C. overnight and counting the next day.

Cell proliferation and TUNEL assays—Proliferating cells were firsttreated with or without PLATINUM-401 for up to 48 h. A portion of cellswere harvested for BrdU incorporation following the manufacturer's (BDBiosciences Pharmingen, San Diego, Calif.) instructions and analyzed byflow cytometry. Harvested cells were also analyzed for apoptosis viadetection of TdT-mediated dUTP nick-end labeling (TUNEL) assay usingApoptosis Detection Systems Fluorescein according to the manufacturer's(Roche, Indianapolis, Ind.) instructions.

Oligonucleotides and plasmids Transfections. The Stat3 antisense(5′-GCTCCAGCATCTGCTGCTTC-3′) (SEQ ID NO:3) or control mismatcholigonucleotides (5′-GCTCCAATACCCGTTGCTTC-3′) (SEQ ID NO:4) weresynthesized using phosphorothioate chemistry and were synthesized with2′-Omethoxyethyl modification of the five terminal nucleotides(underlined; (Mora et al., 2002; Karras et al., 2000)) to increasestability. Transfections of Stat3AS and plasmids were carried out withLipofectamine 2000 (LF) according to the manufacturer's instructions(Invitrogen, Carlsbad, Calif.). Briefly, cells were seeded at 1-2×10⁶cells/10-cm tissue-culture plates 18 h before transfection. Immediatelybefore transfection, cells were washed once with PBS. Cells weretransfected with luciferase reporters (4 μg) in the presence or absenceof v-Src (4 μg), or were transfected with Stat3β (4 μg) or pRC/CMV Stat3Flag (4 μg). Stat3AS transfections were carried out LF alone, or withLF/Stat3 antisense oligonucleotides, or LF/Stat3 mismatcholigonucleotides (final concentration of the oligonucleotides was 250nM). After 2-3 h, the transfection medium was aspirated and cells washedwith PBS before fresh medium added containing 10% FBS was added.Forty-eight h after transfection cells were washed and cytosolic lysatesprepared for luciferase, as previously described (Turkson et al., 1998;Turkson et al., 1999), or processed for TUNEL staining.

Immunohistochemistry—The indirect peroxidase-antiperoxidase test wasperformed on cytospins prepared from cell lines (control and treated).After inhibition of endogenous peroxidase with 0.3% H₂O₂ and methanolfor 30 min, slides were rinsed in PBS [pH 7.4], treated for 30 min with1.5% normal goat serum and then incubated for 1 h with primary antibodyagainst Ki-67 (Vector Laboratories, Burlingame, Calif.) at 1:100dilution. Slides were then rinsed in PBS and incubated with biotinylatedsecondary antibody (Vector Laboratories) at 1:200 dilution for 30 min.Following washing with PBS, the preparations were further incubated inavidin-peroxidase conjugate (Vector Laboratories). The visualization wascarried out with 3,3′-diaminobenzidine (Vector Laboratories) for 2 min.For microscopic evaluation, the preparations were counterstained withhematoxylin and mounted. Negative controls consisted of replacement ofthe primary antibody with PBS. The presence of Ki-67 nuclear stainingwas calculated as percent positive tumor cells in relation to the totalnumber of cells.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Following are examples that illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Identification of PLATINUM-401 as an Inhibitor of Stat3DNA-binding Activity

Compounds from the NCI 2000 diversity set were screened for inhibitionof Stat3 signaling in an in vitro DNA-binding activity assay based onanalysis by electrophoretic mobility shift assay (EMSA). A platinum (IV)complex, PLATINUM-401 (NSC 295558) (FIG. 1E), was identified as a potentinhibitor of Stat3 and was further characterized for its anti-Stat3properties. In the in vitro DNA-binding activity assay, nuclear extractsof equal total protein containing active Stat1, Stat3 and Stat5 werepre-incubated with different concentrations of PLATINUM-401 for 30 minprior to incubation with a [³²P]-labeled oligonucleotide, the m67 highaffinity sis-inducible element (hSIE) probe that binds Stat1 and Stat3,or the mammary gland factor element (MGFe) that binds Stat1 and Stat5.Samples were then subjected to EMSA. Results show that the presence ofPLATINUM-401 in nuclear extracts leads to a dose-dependent reduction inthe levels of DNA-binding activity of Stat3:Stat3 homodimer (FIG. 1A(i), upper band), Stat1:Stat3 heterodimer (FIG. 1A (i), intermediateband), and to a lesser extent of Stat1:Stat1 homodimer (FIG. 1A (i) and(ii), lower band). In contrast, EMSA analysis shows that the presence ofPLATINUM-401 does not significantly affect the level of DNA-bindingactivity of Stat5:Stat5 dimer (FIG. 1A (ii), upper band). These resultsindicate that PLATINUM-401 selectively disrupts Stat3 over Stat1 (IC₅₀values of 1.4 μM and 4.1 μM, respectively, FIG. 1A (iii)), consistentwith our previous findings with other platinum (IV) complexes (Turksonet al., 2004b).

In control studies, the effect of PLATINUM-401 was evaluated on theDNA-binding activity of the E2F1 protein that is unrelated to STATs.Analysis by EMSA shows that pre-incubation with PLATINUM-401 of celllysates containing E2F1 prior to incubation with a [³²P]-labeleddihydrofolate reductase (DHFR) promoter sequence as probe has nosignificant effect on the DNA-binding activity (FIG. 1B (i)). In anothercontrol study, Cisplatin was similarly evaluated on DNA-bindingactivities of STAT proteins and E2F1 and has no detectable effect on theDNA-binding activities of Stat1, Stat3 and Stat5 (FIG. 1B (ii)) and datanot shown), or of E2F1 (data not shown) DNA-binding activity in vitro.These findings together suggest that the effect of PLATINUM-401 isselective for Stat3, and that it is not a general phenomenon of allplatinum compounds to inhibit the activities of transcription factors.

EXAMPLE 2 Lack of Effect of PLATINUM-401 on STAT Proteins Pre-bound toDNA

To further characterize the disruption of in vitro Stat3 DNA-bindingactivity by PLATINUM-401, the sequence of addition of reagents duringthe DNA-binding activity assay was changed to determine how it affectsthe kinetics of PLATINUM-401-mediated inhibition. Nuclear extractscontaining activated Stat3 were first incubated with radiolabeled hSIEprobe for 30 min (to allow prior Stat3 binding to the oligonucleotideprobe) followed by the addition of PLATINUM-401 for an additional 3-30min, and then subjected to EMSA analysis. PLATINUM-401 fails to disruptStat3 DNA-binding activity when the protein is first bound to theDNA-response element probe (FIG. 1C (i), lanes 1-6 compared to controllanes 7-12). These findings indicate that DNA-bound Stat3 protein isoccluded from inhibition by PLATINUM-401, suggesting that the Stat3region required for interaction with PLATINUM-401 is already bound toDNA. By contrast, EMSA analysis shows that the simultaneous addition ofPLATINUM-401 and the hSIE oligonucleotide probe to nuclear extractscontaining activated Stat3 protein results in inhibition of Stat3DNA-binding activity (data not shown), as observed in FIG. 1A,suggesting that Stat3 has a higher preference for PLATINUM-401 overhSIE.

EXAMPLE 3 Interaction of PLATINUM-401 with STAT Proteins

Because the evidence suggested a possible interaction of activated Stat3dimers with PLATINUM-401, the inventors asked the question whetherinactive STAT monomer proteins could do the same. To address this, celllysates of inactive STAT monomer proteins were added to that ofactivated Stat3 and the mixture was pre-incubated with PLATINUM-401 for30 min prior to incubation with radiolabeled hSIE probe and EMSAanalysis. While inactive STAT monomer proteins do not bind the DNAresponse element or alter the binding of activated Stat3 dimer proteinto DNA (FIG. 1C (ii), lane 7), if they could interact with PLATINUM-401the inventors reasoned that they would lower the concentration of thePLATINUM-401 and thereby diminish the extent of PLATINUM-401-mediatedinhibition on the DNA-binding activity of activated Stat3 dimer.

Consistent with this possibility, EMSA analysis shows that the presenceof inactive Stat3 monomer significantly diminishes the inhibitoryeffects of PLATINUM-401 on activated Stat3 DNA-binding activity (FIG. 1C(ii), compare lanes 10-13 to 2-5). Indeed, the presence of the inactiveStat3 monomer protein completely restores the DNA-binding activity ofactive Stat3 dimers that was hitherto abolished by the 1-10 μMPLATINUM-401 (FIG. 1C (ii), lanes 11-12 vs. 3-40) and results in partialrecovery of the active Stat3 DNA-binding activity that is otherwisecompletely disrupted at high (30 μM) concentrations of PLATINUM-401(FIG. 1C (ii), lanes 13 vs. 5). However, at the level of protein presentin the mixture, the inactive Stat3 monomer is insufficient to impacthigher concentrations (50 μM) of PLATINUM-401 and hence no recovery ofactive Stat3 DNA-binding activity is observed (FIG. 1C, last lane).These findings together show that monomer Stat3 interacts withPLATINUM-401 and titrates it out, thereby reducing the levels that areavailable to inhibit active Stat3. The interaction of PLATINUM-401 withStat3 protein is independent of the activation status of the protein.

Similar observations were made when inactive Stat1 monomer was presentin the mixture (FIG. 1C (iii), compare lanes 8-12 to lanes 1-6),suggesting that PLATINUM-401 also interacts with Stat1 protein. Incontrast, inactive Stat5 monomer or a non-STAT-related protein, E2F1,failed to significantly influence PLATINUM-401's effect on Stat3DNA-binding activity (FIG. 1C (iv), compare lanes 8-14 to lanes 1-6 andFIG. 1C (v), compare lanes 7-12 to lanes 1-6).

To investigate the possibility that PLATINUM-401 might alter theintegrity of the hSIE oligonucleotide probe that is used in theDNA-binding activity studies and thereby inhibit Stat3 binding, theoligonucleotide was first treated with PLATINUM-401 for 30 min.Pre-treated oligonucleotide was then radiolabeled and tested for itsability to bind to activated Stat3 in vitro. EMSA analysis reveals thatthe PLATINUM-401 pre-treated radiolabeled hSIE probe bound activatedprotein similarly to the non-treated oligonucleotide probe (FIG. 1D,lanes 1 vs. 2), indicating that the pre-PLATINUM-401 treatment ofoligonucleotide has no observable effect on the ability of theStat3-responsive DNA sequence to bind to activated Stat3 in vitro. WhilePLATINUM-401 was in direct contact with the oligonucleotide probe duringthe pre-treatment stage and prior to radiolabeling of the probe, it isunlikely to have been retained with the oligonucleotide probe followingpurification of the probe and therefore would not have come into contactwith Stat3 protein at the time of the in vitro DNA-binding assay. Thus,under the conditions of the in vitro DNA-binding assay, PLATINUM-401does not directly alter the binding properties of the Stat3-responsiveDNA element, suggesting the DNA element may not directly interact withthe PLATINUM-401.

EXAMPLE 4 Kinetics of PLATINUM-401-mediated Inhibition of Stat3DNA-binding Activity

To further explore the interaction of PLATINUM-401 with Stat3 protein,the levels of activated Stat3 protein and radiolabeled hSIEoligonucleotide probe were varied during the in vitro DNA-binding assayand the extent of PLATINUM-401-mediated inhibition was determined. EMSAanalysis shows that in vitro Stat3 DNA-binding activity increases withincreasing protein amounts (FIG. 2A, lanes 1, 6 and 11). At low (1 μg)protein amount, the presence of PLATINUM-401 causes a strong and adose-dependent decrease in the level of Stat3 DNA-binding activity (FIG.2A, lanes 2-5), consistent with FIG. 1A (i). In contrast, there isdiminished effect of PLATINUM-401 on Stat3 DNA-binding activity athigher (2-3 μg) protein levels (FIG. 2A lanes 6-15), suggesting thatincreasing the Stat3 protein amount overcomes the inhibitory effects ofPLATINUM-401 (FIG. 2A, compare lanes 8-10, 13-15 to lanes 3-5). Theapparent restoration of Stat3 DNA-binding activity at greater proteinamounts suggests high levels of residual active Stat3 protein (in excessof the amount that interacted with PLATINUM-401), which in turn boundthe probe. Altogether, the findings (FIGS. 1 and 2A) indicate thatPLATINUM-401 interacts with Stat3 protein and thereby inhibits Stat3DNA-binding activity.

For the same protein amount, results further show that increasing thelevel of radiolabeled hSIE probe results in an increased level of invitro DNA-binding activity of Stat3 (FIG. 2B, FIG. 2C, lanes 1, 6, 11and 16, and FIG. 2D), until a maximum DNA-binding activity is reached athigher levels of hSIE (a saturation or plateau phase in the plot ofStat3-DNA complex versus amount of hSIE) (FIG. 2D). In the presence ofPLATINUM-401, however, the maximum binding levels (plateau phase)attained are diminished, particularly at high (1-2 μM) concentrations ofPLATINUM-401 (FIG. 2B, FIG. 2C and FIG. 2D), suggesting that increasingprobe levels fail to overcome the inhibitory effect of higherconcentrations of PLATINUM-401. Instead, varying degrees of saturationof Stat3 DNA-binding activity are observed with increasing probe levelsunder high (0.3-2 μM) PLATINUM-401 concentrations (FIG. 2D). Thus,increasing the oligonucleotide probe levels does not restore the maximumDNA-binding activity that is expected for any given amount of protein atdifferent PLATINUM-401 concentrations (1 μM and higher) (FIG. 2D). ALineweaver-Burke (double reciprocal) plot of Stat3-DNA complex versusconcentration of hSIE suggests the inhibitory effect on Stat3DNA-binding activity by PLATINUM-401 displays non-competitive typekinetics (FIG. 2E), with apparent changes in affinity and maximumbinding levels.

EXAMPLE 5 PLATINUM-401 Selectively Blocks Intracellular Stat3 Signalingand Stat3-Mediated Transformation

To further investigate the activities of PLATINUM-401, the inventorstreated malignant cells and measured effects on Stat3 signaling. Instable cell lines (NIH3T3/v-Src/pLucTKS3 and NIH3T3/v-Src/RLSRE)harboring constitutively-active Stat3 and overexpressing Stat3-dependentfirefly (pLucTKS3) and Stat3-independent renilla (pRLSRE) luciferasereporters (Turkson et al., 2001), PLATINUM-401 strongly suppresses theinduction of Stat3-dependent reporter but not the Stat3-independentluciferase reporter or the induction of the β-galactosidase (β-gal) inthe v-Src-transformed fibroblasts expressing β-gal vector (FIG. 3).Similar results were obtained in transient transfection studies of mouseNIH3T3 fibroblasts with the Stat3 reporter, pLucTKS3, or theStat3-dependent VEGF promoter-driven luciferase reporter (pGL2-VEGF-Luc)(Niu et al., 2002) following the activation of Stat3 by v-Src (Turksonet al., 1998) and the treatment with PLATINUM-401 (FIG. 3B, upper twopanels). Taken together with the results in FIG. 1, these findingsindicate that PLATINUM-401 blocks the binding of activated Stat3 to itsresponsive elements in the promoters of target genes. Moreover, in atime- and dose-dependent manner, PLATINUM-401 inhibits the constitutiveor ligand-induced activation of Stat3 DNA-binding activity (FIG. 3C), aswell as tyrosine phosphorylation in mouse fibroblasts (FIG. 3D and datanot shown). The inventors noted IL-6-induced Stat3 activation wasinhibited by PLATINUM-401 as early as 30 min (FIG. 3C, lower panel),while the inhibition of constitutively-active Stat3 in v-Src-transformedNIH3T3/v-Src by PLATINUM-401 required longer than 12 h to be significant(data not shown). Changes in Stat3 protein levels were minimal. Incontrast, the platinum compound has no inhibitory effects on theinduction of Stat3-independent NFκB promoter-driven (NFκB-Luc) or c-fospromoter-driven (pLucSRE) luciferase reporter activity in transienttransfection studies in mouse fibroblasts (FIG. 3B, lower two panels).Results together indicate that at the 5-10 μM PLATINUM-401 that inhibitsintracellular Stat3 signaling, there is no observable non-specificeffect on non-Stat3 related pathways investigated here. These findingstogether demonstrate that PLATINUM-401 selectively represses thetyrosine phosphorylation and DNA-binding activity of Stat3, as well asthe transcriptional regulation in cells by Stat3.

PLATINUM-401 was next evaluated for effects on Stat3-mediated v-Srctransformation (Turkson et al., 1998; Bromberg et al., 1998; Turkson etal., 1999). In soft-agar growth assays of v-Src-transformed(NIH3T3/v-Src) fibroblasts, results show that treatment of cells withPLATINUM-401 strongly suppresses growth (FIG. 4, left panel). Incontrast, similar treatment of v-Ras-transformed (NIH3T3/v-Ras)fibroblasts growing in soft agar only partially inhibits growth (FIG. 4,right panel). These findings are consistent with inhibition ofconstitutively-active Stat3 by PLATINUM-401 and attenuation of growth ofv-Src transformed cells, and show that the PLATINUM-401 inhibitoryeffect is selective against cells harboring constitutively-active Stat3.

EXAMPLE 6 PLATINUM-401-induced Block of Cell Cycle Progression andProliferation

The inventors further examined the biological effects of PLATINUM-401and determined whether any changes might correlate with the inhibitionof constitutively-active Stat3. Normal NIH3T3 and theirv-Src-transformed counterparts, human breast cancer cell lines harboringconstitutively-active Stat3 (MDA-MB-435, MDA-MB-231, and MDA-MB-468) andthose that do not harbor Stat3 activity (MDA-MB-453 and MCF-7), as wellas a human non-small cell lung cancer cell line (A549), a human prostatecancer cell line (DU145) and a murine multiple myeloma cell line(5TGM1), all of which harbor constitutively-active Stat3 (Garcia et al.,2001; Mora et al., 2002; Song et al., 2003), were treated with orwithout PLATINUM-401 for 24 h. Cells were then harvested for nuclearextract preparation and in vitro Stat3 DNA-binding activity assay withEMSA analysis, or processed for cell proliferation and cell cycleanalysis by flow cytometry.

EMSA analysis of Stat3 DNA-binding activity in nuclear extracts preparedfrom malignant cells harboring constitutively-active Stat3 and treatedwith PLATINUM-401 reveals significant inhibition of constitutiveactivation of Stat3 in those cells (FIG. 5A). These observations supportthe inhibition of Stat3 transcriptional activity (FIG. 3) and togetherindicate that PLATINUM-401 selectively blocks constitutive activation ofStat3 signaling in diverse cell types. Normal NIH3T3 fibroblasts andhuman the breast cancer cell line, MDA-MB-453, do not harborconstitutively-active Stat3 (FIG. 5A).

Changes in cell proliferation induced by treatment with PLATINUM-401were assayed in terms of Ki67 proliferation index byimmunohistochemistry. The presence of Ki-67 nuclear staining wascalculated as the percent positive tumor cells in relation to the totalnumber of cells. In contrast to the lack of effect of PLATINUM-401 onthe proliferation of normal NIH 3T3 fibroblasts or the human breastcancer cell line MDA-MB-453 that do not harbor persistent Stat3activity, treatment with PLATINUM-401 causes significant decreases inKi67 nuclear staining for the malignant cells harboringconstitutively-active Stat3 (FIG. 5B), which correlates with inhibitionof Stat3 activity (FIG. 5A).

In flow cytometric analyses for cell cycle changes inPLATINUM-401-treated and untreated (control) cells exposed to BrdU,results show that the breast cancer cell line, MDA-MB-231, is arrestedat G₀/G₁ phase following PLATINUM-401 treatment (FIG. 6). A significantdecrease in S phase cells is also observed, which parallels the drop inDNA synthesis as judged from the level of incorporation of BrdU, andpersists up to 48 h. Similar results are observed after 6 h ofPLATINUM-401-treatment of the breast cancer cell line, MDA-MB-468 (FIG.6, and data not shown). In the case of the MDA-MB-435 cell line, similarobservations are made following 24 h treatment (FIG. 6). In contrast, nosignificant change in cell cycle profile is observed when the breastcancer cell line MDA-MB-453 that does not harbor constitutively-activeStat3 is treated with PLATINUM-401 (FIG. 6). These findings togethershow that malignant cells harboring persistently-elevated Stat3 activityare more sensitive to PLATINUM-401 than cells that do not, consistentwith the ability of PLATINUM-401 to inhibit constitutively-active Stat3and its biological functions.

EXAMPLE 7 PLATINUM-401-mediated Apoptosis of Malignant Cells

Malignant cells harboring persistent Stat3 signaling and those that donot were examined for evidence of apoptosis following treatment withPLATINUM-401. Cells were analyzed for DNA-strand breaks by TUNELstaining Significant TUNEL staining was detected in v-Src-transformedfibroblasts (NIH3T3/v-Src), human breast carcinoma cell lines(MDA-MB-435, MDA-MB-468, MDA-MD-231), human non-small cell lung cancercell line (A549), human prostate carcinoma cell line (DU145), multiplemyeloma cell lines 5TGM1 (mouse) and U266 (human), mouse melanoma cellline (B16), and human pancreatic cancer cell line (Panc1), all of whichharbor constitutively-active Stat3, following treatment withPLATINUM-401 (FIG. 7). In contrast, no TUNEL staining was observed incontrol (DMSO-treated) cells, or in mouse fibroblast cells (NIH3T3) andhuman breast cancer cells (MDA-MB-453) that do not contain aberrantStat3 activity and were treated with PLATINUM-401 (FIG. 7). Theincidence of apoptosis correlated with the prevalence ofconstitutively-active Stat3 in malignant cells (FIG. 7, lower panel).Moreover, the incidence of apoptosis by PLATINUM-401 in breast cancercell lines (MDA-MB-468 and MDA-MB-435) harboring constitutively-activeStat3 compares favorably to that induced by other previouslyinvestigated Stat3 inhibitory approaches, such as Stat3β (Garcia et al.,2001), Stat3 antisense (Stat3AS) (Mora et al., 2002), peptidomimeticinhibitor of Stat3 (ISS 610) (FIG. 7B) (Turkson et al., 2004c), andcontrasts with the absence of apoptosis in breast cancer cell line,MDA-MB-453 that lacks constitutively-active Stat3. These resultsindicate differences in sensitivity of cells to PLATINUM-401, which aredependent on the activation status of Stat3 inside cells. Thesusceptible malignant cells are those that harbor constitutively-activeStat3, which undergo cell cycle arrest and apoptosis in response toPLATINUM-401, consistent with previous reports (Catlett-Falcone et al.,1999; Grandis et al., 2000; Garcia et al., 2001; Epling-Burnette et al.,2001; Bowman et al., 2000b; Turkson et al., 2004b; Mora et al., 2002).

EXAMPLE 8 In Vivo Overexpression of Stat3 Protein Affects PLATINUM-401Activity and Effect

To further elucidate the interaction between Stat3 and PLATINUM-401,v-Src-transformed mouse fibroblasts were transfected with Stat3 proteinand then used to investigate PLATINUM-401 activity. The transfection ofwild-type Stat3 into the v-Src-transformed fibroblasts results in higherStat3 DNA-binding activity over that of parentals, as measured by invitro DNA-binding activity and EMSA analysis (FIG. 7C, left panel, lanes3 and 6 versus 1 and 2). This is due to the increased expression ofStat3 protein and the consequent phosphorylation by oncogenic Srctyrosine kinase that is present in these cells. EMSA analysis of nuclearextracts prepared from parental, mock-transfected cells and treated withPLATINUM-401 show significant reduction in constitutively-active Stat3DNA-binding activity (FIG. 7C, left panel, lanes 4 and 5 versus 1 and2), as previously observed (FIG. 3C, top panel, FIG. 5A, lanes 3 and 4).In contrast, EMSA analysis show that cells transfected with exogenouswild-type Stat3 and treated with PLATINUM-401 show no significantreduction in constitutively-active Stat3 DNA-binding activity (FIG. 7Cleft panel, lane 6 versus 3). This finding is consistent with data fromthe in vitro DNA-binding assay (FIG. 1C (ii)) and together indicatesthat the presence of higher Stat3 protein, either as a monomer or dimer,diminishes the relative potency of PLATINUM-401, thus further confirmingthat PLATINUM-401 interacts with Stat3 protein. Moreover, in diminishingthe effect of PLATINUM-401 on Stat3 DNA-binding activity, studies showthat the overexpression of Stat3 in v-Src-transformed mouse fibroblastsminimizes the extent of PLATINUM-401-mediated apoptosis (FIG. 7C, rightpanel, two far right images). Finally, this finding is important insuggesting that the potency of direct inhibitors of Stat3 activation astherapeutics for tumors harboring constitutively-active Stat3 isinfluenced by the expression levels of the Stat3 protein.

EXAMPLE 9 PLATINUM-401 Represses Induction of Bcl-xL and Cyclin D1

To investigate the molecular changes downstream from Stat3 that maycontribute to the biological responses induced by PLATINUM-401, in situdetection and Western blot analyses were performed for cell cycle andapoptosis regulators. Results show that Cyclin D1 was significantlydiminished in v-Src-transformed mouse fibroblasts (NIH3T3/v-Src) and ahuman breast cancer cell line (MDA-MB-435) in response toPLATINUM-401-induced inhibition of Stat3 activation (FIG. 8). Similarobservations were made for the anti-apoptotic Bcl-xL protein in bothmalignant cell lines, which harbor constitutively-active Stat3,following treatment with PLATINUM-401 (FIG. 8). These findings parallelthe cell cycle or proliferation changes and the induction of apoptosisby the PLATINUM-401 treatment (FIGS. 5, 6, and 7), and indicate thatinhibition of constitutively-active Stat3 by PLATINUM-401 blocks CyclinD1 and Bcl-xL induction.

EXAMPLE 10 Synthesis of 10 g Sample of PLATINUM-401

Synthesis of RPM1581. A mixture of piperazine (Fisher/Acros AC13129)(28.1 g, 0.326 mol), and propylene oxide (Fisher/AcrosAC14962-0010)(41.47 g, 0.714 mol) in anhydrous methanol (32 mL) in a 250mL round bottom flask was refluxed under argon for 3.5 h. After coolingto room temperature, the reaction mixture was allowed to stand at roomtemperature for overnight. The precipitate was filtered, washed withcold methanol (20 mL), then diethyl ether (20 mL) providing the product(28.16 g). The filtrate was concentrated and slurried with MeOH,filtered and dried to provide additional pure product (8.97 g). Thesolids were combined to provide RPM1581-BTC3 (37.13 g, 56%) as a whitesolid.

Synthesis of PLATINUM-401. Chloroplatinic acid (H₂PtCl₆, Aldrichcat.#262587) (13.11 mL of 8 wt % in water, 2.56 mmol) was added over 5minutes to RPM1581-BTC 3 (0.518 g, 2.56 mmol) in a flask cooled in anice water bath. The reaction mixture was clear after approximately 1 mLof chloroplatinic acid solution was added (i.e., the RPM1581 dissolved).An orange precipitate formed while the addition of the remaining RPM1581was ongoing. The reaction mixture was stirred in r.t. for another hour.The orange precipitate was filtered and washed with water (20 mL×4). Thesolid was dried under high vacuum (5 torr) at 80° C. for 3 days toprovide pure PLATINUM-401 (1.180 g, 76%) as an orange solid.

PLATINUM-401 batch 5: 2×scale gave (2.72 g, 87%)

PLATINUM-401 batch 6: 4×scale gave (4.66 g, 74%)

PLATINUM-401 batch 7: 4×scale gave (3.74 g, 59%)

PLATINUM-401 Batches 4, 5, 6, 7 were combined, thoroughly mixed, anddried under high vacuum (5 torr)) at 80° C. overnight. ElementalAnalysis: Found C, 19.56%; H, 3.91; Cl, 35.28; N, 4.41. C₁₀H₂₄Cl₆N₂O₂Ptrequires C, 19.62%; H, 3.95; Cl, 34.75; N, 4.58.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims. In addition, anyelements or limitations of any invention or embodiment thereof disclosedherein can be combined with any and/or all other elements or limitations(individually or in any combination) or any other invention orembodiment thereof disclosed herein, and all such combinations arecontemplated with the scope of the invention without limitation thereto.

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We claim:
 1. A method for treating a subject having an oncologicaldisorder associated with aberrant or excessive Stat3 activity orinteraction in a cell, comprising administering an effective amount of acompound having the structure shown in formula I, formula II, or formulaIII:

to the subject, wherein x is a monovalent anion, and wherein theoncological disorder is breast cancer, lung cancer, prostate cancer,multiple myeloma, melanoma, lymphoma, head and neck squamous cellcarcinoma, or pancreatic cancer.
 2. The method of claim 1, wherein themethod further comprises, prior to said administering, determining thatthe oncological disorder is associated with aberrant or excessive Stat3activity or interaction in a cell.
 3. The method of claim 2, whereinsaid determining comprises measuring a level of Stat3 activity orinteraction in a biological sample collected from the subject andcomparing the measured level to a reference level of Stat3 activity orinteraction.
 4. The method of claim 1, wherein the subject is anon-human mammal.
 5. The method of claim 1, wherein the subject ishuman.
 6. A method for inducing apoptosis in a malignant cell aberrantlyor constitutively expressing active Stat3, comprising contacting thecell with an effective amount of a compound having the structure shownin formula I, formula II, or formula III:

wherein x is a monovalent anion, and wherein the cell is a breast cancercell, lung cancer cell, prostate cancer cell, multiple myeloma cell,lymphoma cell, melanoma cell, head and neck squamous carcinoma cell, orpancreatic cancer cell.
 7. The method of claim 6, wherein the methodfurther comprises, prior to said contacting, determining that the cellor a representative cell aberrantly or constitutively expresses activeStat3.
 8. The method of claim 7, wherein said determining comprisesmeasuring a level of Stat3 activity or interaction in the cell orrepresentative cell and comparing the measured level to a referencelevel of Stat3 activity or interaction.
 9. The method of claim 6,wherein said contacting comprises contacting the cell with a compositioncomprising the compound and a carrier, buffer, adjuvant, or acombination of two or more of the foregoing.
 10. The method of claim 6,wherein said contacting is carried out in vitro.
 11. The method of claim6, wherein said contacting is carried out in vivo.
 12. The method ofclaim 11, wherein said contacting comprises administering the effectiveamount of the compound to a human.
 13. The method of claim 1, whereinthe compound has the structure shown in formula (I).
 14. The method ofclaim 1, wherein the compound has the structure shown in formula (II).15. The method of claim 1, wherein the compound has the structure shownin formula (III).
 16. The method of claim 6, wherein the compound hasthe structure shown in formula (I).
 17. The method of claim 6, whereinthe compound has the structure shown in formula (II).
 18. The method ofclaim 6, wherein the compound has the structure shown in formula (III).